Chimeric vsv virus compositions and methods of use thereof for treatment of cancer

ABSTRACT

Methods of treating cancer including administering to a subject with cancer a pharmaceutical composition including an effective amount of a chimeric VSV virus are disclosed. The chimeric viruses are based on a VSV background where the VSV G protein is replaced with one or more heterologous viral glycoproteins. In the most preferred embodiment, the VSV G protein is replaced with the glycoprotein from Lassa virus or a functional fragment thereof. The resulting chimeric virus is an oncolytic virus that is attenuated and safe in the brain, yet still retains sufficient oncolytic activity to infect and destroy cancer cells such glioblastoma, and to generate an immune response against infected cancer cells. Methods of using chimeric viruses as a platform for immunization against other pathogenic microbes are also provided.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grants1R01CA161048 and RO1 CA175577 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention is generally directed to recombinant vesicular stomatitisvirus (VSV) and methods of use thereof to treat cancer, particularlyglioblastoma.

BACKGROUND OF THE INVENTION

Patients diagnosed with glioblastoma (GBM) generally have about a yearto live. There is no cure for this malignant type of brain cancer. Thedisease generally manifests with subtle changes in brain function, andbecomes worse with seizures, loss of sensation or equilibrium, andprogresses to loss of motor function and mental ability, to finally,death. Surgery, focused radiation, and medical treatment may slow thedisease down and delay death by several months, but unfortunately, thisoften comes at the expense of normal function as neurons, glia, andaxonal pathways are damaged by the treatment) Wrensch, et al.,Neuro-oncol., 4:278-299 (2002)).

Surgery is often done primarily to debulk the tumor, thereby temporarilyrestoring function to surrounding brain regions that were compressed bytumor expansion. A successful tumor resection may remove billions oftumor cells. But inevitably, due to its infiltrative nature, tens ofmillions of tumor cells remain within the brain after surgery (Croteau,et al., Cancer Research, 67(6), 2840-2848 (2007)). A critical feature ofGBMs is that tumor cells are invasive, migrating away from the maintumor body, and continuing to divide elsewhere in the brain. This is onereason why approaches to treatment that focus on a tumor mass areultimately not successful at eliminating the disease.

A considerable number of viruses have been tested for potentialoncolytic activity against glioblastoma (Parker, et al.,Neurotherapeutics, 6:558-569 (2009)). These include human pathogenicviruses that were genetically attenuated to render them safe enough forhuman application, for example HSV (Todo T, Front Biosci., 13:2060-2064(2008)) and adenovirus (Nandi, et al., Expert Opin. Biol. Ther.,9:737-747 (2009); Chiocca, et al., Mol. Ther., 16:618-626 (2008)), andviruses based on vaccination strains, including polio (Dobrikova, etal., Mol. Ther., 16:1865-1872 (2008)), measles (Phuong, et al., CancerRes, 63:2462-2469 (2003)), and vaccinia (Lun, et al., Mol. Ther.,18:1927-1936 (2010)), or adeno-associated viral vectors expressingvarious genes (Maguire, et al., Mol. Ther., 16:1695-1702 (2008)).However, HSV and retroviruses can either enter a latent mode andre-emerge later, or can integrate into host chromosomes, enhancing anoncogenic potential. Furthermore, many of these viruses share thepotential problem that most humans have been exposed to these virusesbefore and their efficacy after systemic application may be challengedby pre-existing immunity.

A promising alternative is the use of viruses that are non-humanpathogens but that display a tropism for tumors, as is the case withmyxoma (Lun, et al., Cancer Res, 65:9982-9990 (2005)), Newcastle diseasevirus (Freeman, et al., Mol. Ther., 13:221-228 (2006), or VSV (Stojdl,et al., Cancer Cell., 4:263-275 (2003)). VSV infections do not integrate(and in fact do not even enter the nucleus), and in animals, includinghumans, are eliminated from the body within 1-2 weeks by the systemicimmune system. In regions of Central America where VSV is endemic, localhuman populations are seropositive for VSV, with no obvious link tosubstantive disease (Tesh, et al, 1969). VSV is rare in the US,indicating a very low level of pre-existing immunity. VSV has beenapproved for human clinical trials where VSV is used as a vaccine vectorto immunize people against dangerous viral or bacterial pathogens(Roberts et al, 1999; Rose et al, 2000; Schwartz et al, 2010).

Another type of attenuated VSV, VSV-M51, shows a reduced ability toblock nuclear pores, thereby allowing normal cells to up-regulateantiviral defenses, and has been described as showing an enhanced safetyprofile (Stojdl, et al., Nat. Med., 6:821-825 (2000); Stojdl, et al.,Cancer Cell, 4:263-275 (2003)). However, this attenuated virus can stillgenerate lethal outcomes after CNS injection.

Some oncolytic viruses have been tested in early phase 1 clinicaltrials, but although the viruses were found to be safe, littletherapeutic effect was seen, and then only in a subset of patients(Zemp, et al., Cytokine Growth Factor Rev., 21:103-117 (2010)),underlining the importance of continuing efforts to find more effectiveoncolytic viruses and delivery strategies (Liu, et al., Mol. Ther.,16:1006-1008 (2008)). Accordingly, despite the advances in thedevelopment and use of oncolytic viruses for treatment of cancer, thereremains a need for improved virus and methods of use therefore forsafely and effective treating cancers such as glioblastoma.

Therefore, it is an object of the invention to provide recombinantoncolytic viruses with improved safety and efficacy profiles.

It is a further object of the invention to provide pharmaceuticalcompositions including an effective amount of recombinant oncolyticviruses to treat cancer in a human subject.

It is another object of the invention to provide methods of usingrecombinant oncolytic virus to kill cancer cells.

It is a further object of the invention to increase the body's immuneresponse against cancer cells.

It is a further object to generate a safer virus-based vaccine againstother non-related microbial antigens.

SUMMARY OF THE INVENTION

Methods of treating cancer including administering to a subject withcancer a pharmaceutical composition including an effective amount of achimeric VSV virus are disclosed. The chimeric viruses are based on aVSV background where the VSV G protein is replaced with one or moreheterologous viral glycoproteins. In the most preferred embodiment, theVSV G protein is replaced with the glycoprotein from Lassa virus or afunctional fragment thereof. The Examples below show that replacement ofthe VSV G protein with a heterologous glycoprotein, particularly theglycoprotein from Lassa virus, results in an oncolytic virus that ishighly attenuated and safe in the brain, yet still retains sufficientoncolytic activity to infect and destroy cancer cells such asglioblastoma and intracranial melanoma metastases. The chimeric viruscan be further modified to express one or more therapeutic proteins,reporters, vaccine antigens, or targeting moieties. Exemplarytherapeutic proteins and reporters include, but are not limited to,IL-28, IL-2, FLT3L, GM-CSF, IL-4, IL-7, IL-12, TRAIL, carcinoembryonicantigen, secreted alkaline phosphatase, the beta subunit of chorionicgonadotropin, and green fluorescent protein.

Methods can include administering to a subject an effective amount ofthe virus to reduce one or more symptoms of cancer, for example tumorburden. The cancer can be multiple myeloma, bone, bladder, brain,breast, cervical, colo-rectal, esophageal, kidney, liver, lung,nasopharangeal, pancreatic, prostate, skin, stomach, and uterine. In apreferred embodiment, the methods are used to treat brain cancer andbrain metastases. Brain cancers include, but are not limited to,oligodendroglioma, meningioma, supratentorial ependymona, pineal regiontumors, medulloblastoma, cerebellar astrocytoma, infratentorialependymona, brainstem glioma, schwannomas, pituitary tumors,craniopharyngioma, optic glioma, and astrocytoma. In a particularlypreferred embodiment, the cancer is glioblastoma.

The virus is typically administered in a dosage of between about 10² andabout 10¹² PFU, more preferably between about 10² and about 10¹² PFU.The pharmaceutical composition can be administered locally to the siteof the cancer. For example, the composition can be injected into oradjacent to a tumor in the subject, or via catheter into a tumorresection cavity, for example, by convection-enhanced delivery (CED).The pharmaceutical composition can be administered systemically to thesubject, for example by intravenous, intra-arterial, or intrathecalinjection or infusion.

The virus can be administered in combination with one or more additionaltherapeutic agents. The one or more additional therapeutic agents canbe, for example, an anticancer agent such as a chemotherapeutic agent, atherapeutic protein such as IL-2, or an immunosuppressant. Theimmunosuppressant can be a histone deacetylase (HDAC) inhibitor or aninterferon blocker, for example, valproate, the vacccinia protein B18R,Jak inhibitor 1, or vorinostat, which can be used to reduce or delay thesubject's immune response to the virus.

The pharmaceutical composition can be administered in combination withsurgery. In some embodiments, the subject is pre-treated with animmunizing composition including a virus effective to immunize thesubject to the chimeric VSV virus prior to administration of thepharmaceutical composition. The virus in the immunizing composition canbe the chimeric VSV virus. Immunizing the subject against the virus canincrease the ability of the subject's immune system to clear the virusfollowing therapeutic treatment if needed.

Other methods of treating cancer are also disclosed. For example, amethod of treating a subject for cancer can include (a) infectingisolated cancer cells with an effective amount of a chimeric VSV virusand (b) administrating the infected cells to the subject in an effectiveamount to induce an immune response against the cancer cells in thesubject. In some embodiments, the method includes irradiating the cellsto prevent their proliferation in the subject. The method can be used totherapeutically or prophylactically treat cancer in the subject.

Methods of priming the immune system for attacking cancer cells andadaptive T cell therapy are also disclosed. The priming can occur invitro or in vivo. A particular embodiment of preparing cells foradaptive T cell therapy includes administering to a subject with cancera pharmaceutical composition including an effective amount of a chimericVSV virus to increase the number of cytotoxic T cells (CTL) which candirectly kill the cancer, or to increase the number of CD4+ T and/orCD8+ T cells which can direct an immune response against the cancer. TheT cells can be isolated from the subject and propagated in vitro. The Tcells can be administered back to the same subject, or another subjectin need thereof.

Pharmaceutical dosage units and kits including an effective amount ofthe disclosed chimeric viruses are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a histogram showing percent infected cells (% GFP) for eachof three gliomas tested (U87, U118 and CT2A, are represented bydifferent shading) 24 hours after infection with 0.1 multiplicity ofinfection (MOT) of a VSV ((control) also referred to herein as VSV-wtG),or a chimeric VSV virus wherein the G protein was replaced with theglycoprotein from Lassa (as referred to herein as Lassa-VSV, VSV-LASV-G,and LASV), rabies (also referred to herein as VSV-RABV-G and RABV), LCMV(also referred to herein as VSV-LCMV-G), Ebola (also referred to hereinas EBOV, VSV-EBOV-G, and Ebola-VSV), or Marburg (also referred to hereinas MARV and VSV-MARV-G). FIG. 1B is a histogram showing the viralreplication (Titer, pfu/ml) VSV-wtG (control), or a chimeric VSV viruswherein the G protein was replaced with the glycoprotein from Lassa,rabies, LCMV, Ebola, or Marburg. FIG. 1C is an illustration of therelative mean diameter and the small vertical line surmounting each theSEM, of 60 randomly selected fluorescent plaques measured and normalizedto VSV-wtG plaque size on monolayers of U87, U118 and normal human braincells, 24 hpi with VSV-wtG (control), or a chimeric VSV virus whereinthe G protein was replaced with the glycoprotein from Lassa, LCMV, orEbola. FIG. 1D is a histogram showing the fraction of infected cells ofmouse brain cultures (% gila (top portion of the bar) versus % neurons(bottom portion of the bar)) for cells infected with VSV-wtG (control),or a chimeric VSV virus wherein the G protein was replaced with theglycoprotein from Lassa, rabies, LCMV, Ebola, or Marburg (MOI 5).

FIG. 2A is a Kaplan-Meier survival curve showing the % survival of miceinfected with chimeric Lassa-VSV, chimeric Ebola-VSV, attenuatedVSV-MΔ51, or attenuated VSV-1′GFP over time (in days post-inoculation)following intracranial inoculation with virus. FIG. 2B is a Kaplan-Meiersurvival curve showing the % survival of mice infected with chimericLassa-VSV or VSV-IFN over time (in days post-inoculation) followingintracranial inoculation with virus.

FIG. 3A is a histogram showing infection of human neuronal cultures withVSV-wtG or chimeric Lassa-VSV (GFP expression %) with or withouttreatment with 100 IU/ml of interferon (IFN). FIG. 3B is a Kaplan-Meiersurvival curve showing the % survival of normal mice infected withchimeric Lassa-VSV, and IFNα/β-R knockout (−/−) mice infected withVSV-wtG or Lassa-VSV (in days post-inoculation) following intracranialinoculation with virus. FIG. 3C is a histogram showing virus binding andinternalization (relative expression by qRT-PCR) of VSV-wtG andLassa-VSV in neurons and giloma cells at 4° C. and 37° C. FIG. 3D is adot plot showing the quantification of VSV-wtG viral replication inneurons (▴) and U87 glioma (♦) cells assessed by plaque assay at 15 and24 hpi. FIG. 3E is a dot plot showing the quantification of VSV-LASV-Gviral replication in neurons (▴) and U87 glioma (♦) cells assessed byplaque assay at 15 and 24 hpi.

FIG. 4 is a Kaplan-Meier survival curve showing the % survival of miceinfected with Lassa-VSV (solid line), and Ebola-VSV (dotted line) anduninfected control (solid line), respectively, over time (in days)following intracranial glioma xenograph and subsequent inoculation withvirus. The times of i.c.U87 xenograft, and i.v. virus inoculation areindicated. The increased longevity of tumor bearing mice receivingLassa-VSV is statistically significant (p<0.01; n=8 in each group).

FIG. 5 is a line graph showing the % body weight of mice followingintracranial glioma xenograph and subsequent systemic infection withLassa-VSV, Ebola-VSV and uninfected control, respectively, over time (indays post-inoculation).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, the term “isolated” describes a compound of interest(e.g., either a polynucleotide or a polypeptide) that is in anenvironment different from that in which the compound naturally occurse.g., separated from its natural milieu such as by concentrating apeptide to a concentration at which it is not found in nature.“Isolated” includes compounds that are within samples that aresubstantially enriched for the compound of interest and/or in which thecompound of interest is partially or substantially purified. Withrespect to nucleic acids, the term “isolated” includes anynon-naturally-occurring nucleic acid sequence, since suchnon-naturally-occurring sequences are not found in nature and do nothave immediately contiguous sequences in a naturally-occurring genome.

As used herein, the term “nucleic acid(s)” refers to any nucleic acidcontaining molecule, including, but not limited to, DNA or RNA. The termencompasses sequences that include any of the known base analogs of DNAand RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine. In accordance with standard nomenclature, nucleicacid sequences are denominated by either a three letter, or singleletter code as indicated as follows: adenine (Ade, A), thymine (Thy, T),guanine (Gua, G) cytosine (Cyt, C), uracil (Ura, U).

As used herein, the term “polynucleotide” refers to a chain ofnucleotides of any length, regardless of modification (e.g.,methylation).

As used herein, the term “gene” refers to a nucleic acid (e.g., DNA orRNA) sequence that comprises coding sequences necessary for theproduction of a polypeptide, RNA (e.g., including but not limited to,mRNA, tRNA and rRNA) or precursor. The polypeptide, RNA, or precursorcan be encoded by a full length coding sequence or by any portionthereof. The term also encompasses the coding region of a structuralgene and the sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The term “gene”encompasses both cDNA and genomic forms of a gene, which may be made ofDNA, or RNA. A genomic form or clone of a gene may contain the codingregion interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “nucleic acid molecule encoding,” refers to theorder or sequence of nucleotides along a strand of nucleotides. Theorder of these nucleotides determines the order of amino acids along thepolypeptide (protein) chain. The nucleotide sequence thus codes for theamino acid sequence.

As used herein, the term “polypeptide” refers to a chain of amino acidsof any length, regardless of modification (e.g., phosphorylation orglycosylation). In accordance with standard nomenclature, amino acidresidue sequences are denominated by either a three letter or a singleletter code as indicated as follows: Alanine (Ala, A), Arginine (Arg,R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine. (Cys, C),Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine(His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K),Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine(Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y),and Valine (Val, V).

As used herein, a “variant,” “mutant,” or “mutated” polynucleotidecontains at least one polynucleotide sequence alteration as compared tothe polynucleotide sequence of the corresponding wild-type or parentpolynucleotide. Mutations may be natural, deliberate, or accidental.Mutations include substitutions, deletions, and insertions.

As used herein, a “nucleic acid sequence alteration” can be, forexample, a substitution, a deletion, or an insertion of one or morenucleotides. An “amino acid sequence alteration” can be, for example, asubstitution, a deletion, or an insertion of one or more amino acids.

As used herein, the term “immunizing virus” includes infectious virus,viral subunits, viral proteins and antigenic fragments thereof, nucleicacids encoding viral subunits, antigenic proteins or polypeptides, andexpression vectors containing the nucleic acids.

As used herein, a “vector” is a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. The vectors describedherein can be expression vectors.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” means a dosage sufficient to treat, inhibit, oralleviate one or more symptoms of a disease state being treated or tootherwise provide a desired pharmacologic and/or physiologic effect. Theprecise dosage will vary according to a variety of factors such assubject-dependent variables (e.g., age, immune system health, etc.), thedisease, and the treatment being effected.

As used herein, the, terms “neoplastic cells,” “neoplasia,” “tumor,”“tumor cells,” “cancer” and “cancer cells,” (used interchangeably) referto cells which exhibit relatively autonomous growth, so that theyexhibit an aberrant growth phenotype characterized by a significant lossof control of cell proliferation (i.e., de-regulated cell division).Neoplastic cells can be malignant or benign.

As used herein, an “immunogen” or “immunogenic amount” refers to theability of a substance (antigen) to induce an immune response. An immuneresponse is an alteration in the reactivity of an organisms' immunesystem in response to an antigen. In vertebrates this may involveantibody production, induction of cell-mediated immunity, complementactivation or development of immunological tolerance.

As used herein, an “adjuvant” is a substance that increases the abilityof an antigen to stimulate the immune system.

As used herein, “attenuated” refers to refers to procedures that weakenan agent of disease (a pathogen). An attenuated virus is a weakened,less vigorous virus. A vaccine against a viral disease can be made froman attenuated, less virulent strain of the virus, a virus capable ofstimulating an immune response and creating immunity but not causingillness or less severe illness. Attenuation can be achieved by chemicaltreatment of the pathogen, through radiation, or by geneticmodification, using methods known to those skilled in the art.Attenuation may result in decreased proliferation, attachment to hostcells, or decreased production or strength of toxins.

As used herein, “subject,” “individual,” and “patient” refer to anyindividual who is the target of treatment using the disclosedcompositions. The subject can be a vertebrate, for example, a mammal.Thus, the subject can be a human. The subjects can be symptomatic orasymptomatic. The term does not denote a particular age or sex. Asubject can include a control subject or a test subject.

As used herein, “identity,” as known in the art, is a relationshipbetween two or more polypeptide sequences, as determined by comparingthe sequences. In the art, “identity” also means the degree of sequencerelatedness between polypeptide as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including, but not limited to, thosedescribed in (Computational Molecular Biology, Lesk, A. M., Ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., Ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,Eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073(1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs. Thepercent identity between two sequences can be determined by usinganalysis software (i.e., Sequence Analysis Software Package of theGenetics Computer Group, Madison Wis.) that incorporates the Needelmanand Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST,and XBLAST). The default parameters are used to determine the identityfor the polypeptides of the present disclosure.

By way of example, a polypeptide sequence may be identical to thereference sequence, that is be 100% identical, or it may include up, toa certain integer number of amino acid alterations as compared to thereference sequence such that the % identity is less than 100%. Suchalterations are selected from: at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in the reference polypeptideby the numerical percent of the respective percent identity (divided by100) and then subtracting that product from said total number of aminoacids in the reference polypeptide.

As used herein “pharmaceutically acceptable carrier” encompasses any ofthe standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water and emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents.

As used herein, “treatment” or “treating” means to administer acomposition to a subject or a system with an undesired condition. Thecondition can include a disease. “Prevention” or “preventing” means toadminister a composition to a subject or a system at risk for thecondition. The condition can include a predisposition to a disease. Theeffect of the administration of the composition to the subject (eithertreating and/or preventing) can be, but is not limited to, the cessationof one or more symptoms of the condition, a reduction or prevention ofone or more symptoms of the condition, a reduction in the severity ofthe condition, the complete ablation of the condition, a stabilizationor delay of the development or progression of a particular event orcharacteristic, or minimization of the chances that a particular eventor characteristic will occur. It is understood that where treat orprevent are used, unless specifically indicated otherwise, the use ofthe other word is also expressly disclosed.

II. Compositions

VSV, a member of the Rhabdoviridae family, is enveloped and has anegative-strand 11.2-kb RNA genome that comprises five protein-encodinggenes (N, P, M, G, and L) (Lyles, et al., Fields virology, 5^(th) ed.,Lippincott Williams & Wilkins, 1363-1408 (2007)). It is a nonhumanpathogen which can cause mild disease in livestock. Infection in humansis rare and usually asymptomatic, with sporadic cases of mild flu-likesymptoms. VSV has a short replication cycle, which starts withattachment of the viral glycoprotein spikes (G) to an unknown butubiquitous cell membrane receptor. Nonspecific electrostaticinteractions have also been proposed to facilitate viral binding (Lyles,et al., Fields virology, 5^(th) ed., Lippincott Williams & Wilkins,1363-1408 (2007)). Upon internalization by clathrin-dependentendocytosis, the virus-containing endosome acidifies, triggering fusionof the viral membrane with the endosomal membrane. This leads to releaseof the viral nucleocapsid (N) and viral RNA polymerase complex (P and L)into the cytosol.

The viral polymerase initiates gene transcription at the 3′ end of thenon-segmented genome, starting with expression of the first VSV gene(N). This is followed by sequential gene transcription, creating agradient, with upstream genes expressed more strongly than downstreamgenes. Newly produced VSV glycoproteins are incorporated into thecellular membrane with a large extracellular domain, a 20 amino acidtrans-membrane domain, and a cytoplasmic tail consisting of 29 aminoacids. Trimers of G protein accumulate in plasma membrane microdomains,several of which congregate to form viral budding sites at the membrane(Lyles, et al., Fields virology, 5^(th) ed., Lippincott Williams &Wilkins, 1363-1408 (2007)). Most cells activate antiviral defensecascades upon viral entry, transcription, and replication, which in turnare counteracted by VSV matrix protein (M). VSV M protein's multitude offunctions include virus assembly by linking the nucleocapsid with theenvelope membrane, induction of cytopathic effects and apoptosis,inhibition of cellular gene transcription, and blocking of host cellnucleocytoplasmic RNA transfer, which includes blocking of antiviralcellular responses (Ahmed, et al., Virology, 237:378-388 (1997)).

Certain native, engineered, and recombinant VSV strains have been shownto target several tumor types, including gliomas, and give a strongoncolytic action, both in vitro and in vivo (Paglino and van den Pol,2011) (Wollmann, et al, 2005; 2007; 2010; Ozduman et al, 2008). However,there remains a need for improved recombinant VSVs that are bothefficacious for treating cancer and exhibit low pathogenicity to healthyhost cells. This is particularly important in the brain where matureneurons do not replicate, and once lost, are normally not replaced.Although some evidence indicates that attenuated VSVs show reducedneurotoxicity, CNS complications have been difficult to eliminatecompletely (Obuchi et al, 2003; van den Pol et al, 2002; 2009).

It has been discovered that recombinant, chimeric VSV viruses where theG gene is substituted with a gene encoding a heterologous glycoproteinprotein have oncolytic potential in targeting and destroying cancercells with little pathogenicity to healthy host cells. Recombinant VSVviruses, pharmaceutical compositions including recombinant VSV viruses,and methods of use thereof for treating cancer are provided. Asdiscussed in more detail below, preferably, the virus targets and killstumor cells, and shows little or no infection of normal cells.

A. Chimeric G-Gene Substituted VSV Virus

The disclosed viruses are chimeric VSV viruses that are typically basedon a VSV background strain, also referred to herein as a VSV backbone,wherein the G gene is substituted for a heterologous glycoprotein. Asdiscussed in more detail below, the chimeric virus can also includeadditional genetic changes (e.g., additions, deletions, substitutions)relative to the background VSV virus, and can have one or moreadditional transgenes.

1, VSV Background Strain

Useful VSV virus background strains can be viruses that are known in theart, or they can be mutants or variants of known viruses. Any suitableVSV strain or serotype may be used, including, but not limited to, VSVIndiana, VSV New Jersey, VSV Alagoas, (formerly Indiana 3), VSV Cocal(formerly Indiana 2), VSV Chandipura, VSV Isfahan, VSV San Juan, VSVOrsay, or VSV Glasgow. The VSV virus background can be a naturallyoccurring virus, or a virus modified, for example, to increase ordecrease the virulence of the virus, and/or increase the specificity orinfectivity of the virus compared to the parental strain or serotype.The virus can be a recombinant virus that includes genes from two ormore strains or serotypes. For example, the VSV background strain can bea recombinant VSV with all five genes of the Indiana serotype of VSV. Inanother exemplary embodiments, the N, P, M, and L genes originates fromthe San Juan strain, and the G gene from the Orsay strain.

It may be desirable to further reduce the neurovirulence of the virusesused in the disclosed methods, particularly the virulence of thetherapeutic virus, by using an attenuated virus. A number of suitableVSV mutants have been described, see for example (Clarke, et al., J.Virol., 81:2056-64 (2007), Flanagan, et al., J. Virol., 77:5740-5748(2003), Johnson, et al., Virology, 360:36-49 (2007), Simon, et al., J.Virol., 81:2078-82 (2007), Stojdl, et al., Cancer Cell, 4:263-275(2003)), Wollmann, et al., J. Virol, 84(3):1563-73 (2010) (epub 2010),WO 2010/080909, U.S. Published Application No. 2007/0218078, and U.S.Published Application No 2009/0175906.

Recombinant VSVs derived from DNA plasmids also typically show weakenedvirulence (Rose, et al., Cell, 106:539-549 (2001)). Attenuation of VSVvirulence can also be accomplished by one or more nucleotide sequencealterations that result in substitution, deletion, or insertion of oneor more amino acids of the polypeptide it encodes.

In some embodiments, the VSV background strain is a VSV virus modifiedto attenuate virus growth or pathogenicity or to reduce the ability tomake infectious progeny viruses. VSV strains and methods of making suchVSV strains are known in the art, and described in, for example, U.S.Published Application No. 2012/0171246.

For example, one strategy is to attenuate viral pathogenicity byreducing the ability of the virus to suppress host innate immuneresponses without compromising the yield of infectious progeny. This canbe accomplished by mutating the M protein as described, for example, inAhmed, J Viral., 82(18):9273-9277 (2008). The M protein is amultifunctional protein that is involved in the shutoff of hosttranscription, nuclear cytoplasmic transport, and translation duringvirus infection (Lyles, Microbial. Mol. Biol. Rev. 64:709-724 (2000)).Mutation and/or deletion of one or more amino acids from the M protein,for example MΔ51, or M51A mutants can result in viral protein that isdefective at inhibiting host gene expression. It may also be desirableto switch or combine various substitutions, deletions, and insertions tofurther modify the phenotype of the virus. For example, the recombinantVSV background can have a deletion or mutation in the M protein.

Altering the relative position of genes can also be used to attenuatevirus (Clarke, et al., J. Virol., 81:2056-2064, (2007), Cooper, et al.,J. Virol., 82:207-219 (2008), Flanagan, et al., J. Virol., 75:6107-6114(2001)). VSV is highly immunogenic, and a substantial B and T cellresponse from the adaptive immune system will ultimately limit VSVinfection, which will halt long-lasting viral infections. A virus thatshows enhanced selectivity, and a faster rate of infection, will have agreater likelihood of eliminating cancer cells before the virus iseliminated by the immune system. However, the use of VSV against cancercells does not have to be restricted to a single application. Bymolecular substitution of the G-protein for enhancing immune responsesagainst foreign genes expressed by VSV, one could switch the original Gprotein of the virus (e.g., Indiana VSV) with the G protein from anotherstrain or serotype (e.g., VSV New Jersey or Chandipura), allowing aslightly different antigen presentation, and reducing the initialresponse of the adaptive immune system to second or third oncolyticinoculations with VSV.

Therefore, the disclosed chimeric viruses can have a VSV genome that isrearranged compared to wildtype VSV. For example, shifting the L-gene tothe sixth position, by rearrangement or insertion of an additional geneupstream, can result in attenuated L-protein synthesis and a slightreduction in replication (Dalton and Rose, Virology, 279(2):414-21(2001)), an advantage when considering treatment of the brain.

Repeat passaging of virulent strains under evolutionary pressure canalso be used to generate attenuated virus, increase specificity of thevirus for a particular target cell type, and/or increase the oncolyticpotential of the virus. For example, VSV-rp30 (“30 times repeatedpassaging”) is a wild-type-based VSV with an enhanced oncolytic profile(Wollmann, et al., J. Virol. 79:6005-6022 (2005)). As described in WO2010/080909, VSV-rp30 has a preference for glioblastoma over controlcells and an increased cytolytic activity on brain tumor cells.Accordingly, in some embodiments, the VSV background of the disclosedchimeric viruses is one that has been modified to attenuate the virus,increase specificity of the virus for a particular target cells, and/orincrease the oncolytic potential of the virus relative to a wildtype orstarting stain.

2. Heterologous Glycoproteins

The disclosed chimeric VSV viruses have a heterologous glycoprotein.Typically, the disclosed chimeric VSV viruses are viruses that lack theG protein of VSV. Instead the chimeric VSV viruses have a glycoprotein(e.g., G protein or GP protein) from a distinct, non-VSV virus.

As demonstrated in the Examples below, glycoproteins for a number ofdifferent viruses can be substituted into a VSV background to create achimeric VSV that can infect cancer cells. Suitable glycoproteins can befrom, for example, Lassa, rabies, lymphocytic choriomeningitis virus(LCMV), Ebola, or Marburg virus. The Examples below show that anEbola-VSV, and even more so a Lassa-VSV chimera, are particularlyeffective at killing brain cancers with little or no toxicity to healthyor normal cells. Other viral glycoprotein such as those from rabies,lymphocytic choriomeningitis virus (LCMV), or Marburg virus may be moresuitable for targeting other cancer types, such as one or more of thecancers discussed in more detail below. It is believed that VSV chimericviruses including an LCMV glycoprotein in place of the VSV glycoproteinmay show some advantages over the VSV glycoprotein in infecting somecancer or sarcoma cells with enhanced innate immunity, such as thevirus-resistant sarcoma cells described in Paglino and van den Poi, J.Virol., 85:9346-9358 (2011). In some embodiments, the G protein in theVSV chimeric virus is a heterologous G, wherein the G protein is not a Gprotein from LCMV.

In place of the Lassa glycoprotein which has a broad spectrum of cellsto which it binds, the VSV chimeric virus can have a glycoprotein fromanother arena virus. Other arenaviruses may have the same, similar, ordifferent cellular binding receptors to Lassa. In some embodiments, theglycoprotein is a viral glycoprotein, preferably an arenavirusglycoprotein, that binds to one or more of the same cell receptors asLassa glycoprotein. In some embodiments, the glycoprotein is a viralglycoprotein, preferably an arenavirus glycoprotein that binds to one ormore similar cell receptors as Lassa glycoprotein. In some embodiments,the glycoprotein is an areanvirus glycoprotein that binds to differentcell receptor(s) than Lassa glycoprotein. Such chimeric viruses may alsobe safe viruses for use in oncolysis or as vaccine vectors. Exemplaryarenaviruses include, but are not limited to, Old World complexarenaviruses such as Kodoko, Lujo, Mobala, Dank, Gbagroube, Ippy, MerinoWalk, Menekre, Mobala, and Mopeia, and New World arenaviruses such asGuanarito, Junin, Machupo, Sabia, Whitewater arroyo, Parana, Tamiami,Latino, plexal, and Chapare. New World arenavirus glycoproteins maytarget receptors different that those targeted by the Lassaglycoprotein.

a. Lassa G proteins

In the most preferred embodiment, the G protein of VSV is substitutedwith a glycoprotein from a Lassa virus. Lassa virus is an Arenavirus.The genomic structure or Arenaviruses and the genetic diversity of Lassavirus strains are discussed in Bowen, et al., J. Virology, 6992-7004(2000). Viruses of the genus Arenavirus, family Arenaviridae, areenveloped viruses with a genome consisting of two single stranded RNAspecies designated small (S) and large (L). Each segment contains twonon-overlapping genes arranged in an ambisense orientation. The viralpolymerase (L protein) gene is encoded at the 3′ end of the L RNA in thegenome-complementary sense, whereas the Z protein is encoded at the 5′end of the L RNA in the genomic sense. In a similar fashion, thenucleoprotein (NP) gene is encoded at the 3′ end of the S RNA, whereasthe glycoprotein precursor (GPC) is encoded at the 5′ end of the S RNA.The GPC is post-translationally cleaved into the envelope glycoproteinsGP1 and GP2. The arenaviruses have been divided into two groups, the NewWorld arenaviruses and the Old World arenaviruses. Lassa virus is an OldWorld arenavirus.

The glycoprotein can come from any Lassa virus. The Lassa virusglycoprotein can be from a naturally occurring virus, or a virusmodified, for example, to increase or decrease the virulence of thevirus, and/or increase the specificity or infectivity of the viruscompared to the parental strain or serotype. Suitable strains andserotypes of Lassa virus from which the glycoprotein of the chimeric VSVvirus can be derived are known in the art and include, for example,fifty-four strains identified and characterized in Bowen, et al., J.Virology, 6992-7004 (2000). Common Lassa virus stains include Lassavirus strain 803213, Lassa virus strain Acar 3080, Lassa virus strainAV, Lassa virus strain Josiah, Lassa virus strain LP, Lassa virus strainMacenta, Lassa virus strain NL, Lassa virus strain Pinneo, Lassa virusstrain Weller, and Lassa virus strain Z148.

Preferably, the chimeric virus's genome, or plasmid(s) encoding thevirus's genome encode the entire Lassa virus glycoprotein precursor(GPC), such that both GP1 and GP2 are expressed and contribute toformation of the chimeric virus's envelope. In some embodiments, thechimeric virus's genome, or plasmid(s) encoding the virus's genomeencode less than the entire Lassa virus glycoprotein precursor (GPC).For example, in some embodiments, the viral genome or plasmid(s)encoding recombinant viral genome encodes a glycoprotein that is atruncated GPC, or only GP1 or only GP2.

The glycoprotein can be from Lassa strain Josiah. In a particularembodiment, the chimeric viral genome includes the nucleic acid sequence

(SEQ ID NO: 1   1 cgcaccgggg atcctaggca tttttggttg cgcaattcaa gtgtcctatt taaaatggga  61 caaatagtga cattcttcca ggaagtgcct catgtaatag aagaggtgat gaacattgtt 121 ctcattgcac tgtctgtact agcagtgctg aaaggtctgt acaattttgc aacgtgtggc 181 cttgttggtt tggtcacttt cctcctgttg tgtggtaggt cttgcacaac cagtctttat 241 aaaggggttt atgagcttca gactctggaa ctaaacatgg agacactcaa tatgaccatg 301 cctctctcct gcacaaagaa caacagtcat cattatataa tggtgggcaa tgagacagga 361 ctagaactga ccttgaccaa cacgagcatt attaatcaca aattttgcaa tctgtctgat 421 gcccacaaaa agaacctcta tgaccacgct cttatgagca taatctcaac tttccacttg 481 tccatcccca acttcaatca gtatgaggca atgagctgcg attttaatgg gggaaagatt 541 agtgtgcagt acaacctgag tcacagctat gctggggatg cagccaacca ttgtggtact 601 gttgcaaatg gtgtgttaca gacttttatg aggatggctt ggggtgggag ctacattgct 661 cttgactcag gccgtggcaa ctgggactgt attatgacta gttatcaata tctgataatc 721 caaaatacaa cctgggaaga tcactgccaa ttctcgagac catctcccat cggttatctc 781 gggctcctct cacaaaggac tagagatatt tatattagta gaagattgct aggcacattc 841 acatggacac tgtcagattc tgaaggtaaa gacacaccag ggggatattg tctgaccagg 901 tggatgctaa ttgaggctga actaaaatgc ttcgggaaca cagctgtggc aaaatgtaat 961 gagaagcatg atgaggaatt ttgtgacatg ctgaggctgt ttgacttcaa caaacaagcc1021 attcaaaggt tgaaagctga agcacaaatg agcattcagt tgatcaacaa agcagtaaat1081 gctttgataa atgaccaact tataatgaag aaccatctac gggacatcat gggaattcca1141 tactgtaatt acagcaagta ttggtacctc aaccacacaa ctactgggag aacatcactg1201 cccaaatgtt ggcttgtatc aaatggttca tacttgaacg agacccactt ttctgatgat1261 attgaacaac aagctgacaa tatgatcact gagatgttac agaaggagta tatggagagg1321 caggggaaga caccattggg tctagttgac ctctttgtgt tcagtacaag tttctatctt1381 attagcatct tccttcacct agtcaaaata ccaactcata ggcatattgt aggcaagtcg1441 tgtcccaaac ctcacagatt gaatcatatg ggcatttgtt cctgtggact ctacaaacag1501 cctggtgtgc ctgtgaaatg gaagagatga gacccttgtc agggcccccg tgacccaccg1561 cctattggcg gtgggtcacg ggggcgtcca tttacagaac gactctaggt gtcgatgttc1621 tgaacaccat atctctgggc agcactgctc tcaaaaccga tgtgttcagt cctcctgaca1681 ctgctgcatc aaacatgatg cagtccatta gtgcacagtg aggggttatt tcctctttac1741 cgcctctttt cttcttttca acaacgacac ctgtgtgcat gtggcataag tctttatact1801 ggtcccagac tgcattttca tacttcctgg aatcagtttt gctgagggca atatcaatta1861 gtttaatgtc ttttcttcct tgtgattcaa ggagtttcct tatgtcatcg gacccctgac1921 aggtaatgac catattccgg gggagtgcat caatgacagc actggtcaag cccggttgtg1981 tagcgaagag gtctgtgaca tcaatcccat gtgagtactt agcatcctgc ttgaactgct2041 ttaaatcagt aggttcacgg aagaagtgta tgtagcagcc tgaacttggt tgatagaggg2101 caatttccac tggatcttca ggtcttcctt caatgtccat ccaggtctta gcatttgggt2161 caagttgcag cattgcatcc ttgagggtaa tcagctgaga ataggtaagc ccagcggtaa2221 accctgccga ctgcagggat ttactggaat tgttgctgtc agctttctgt ggcttcccat2281 ctgattccag atcaacgaca gtgttttccc aggcccttcc tgttattgag gttcttgatg2341 caatatatgg ccatccatct cctgacaaac aaatcttgta gagtatgttt tcataaggat2401 tcctttcacc aggggtgtct gaaatgaaca ttccaagagc cttcttgacc tttaaaatgg2461 atttgaggat accatccatt gtctgaggtg acaccttgat tgtctccaac atattgccac2521 catccagcat gcaagctcct gccttcacag ctgcacccaa gctaaaatta taacctgaga2581 tattcaaaga gcttttcttg gtgtcaatca tatttaggat gggatgactt tgagtcagcc2641 tgtctaagtc tgaagtgttg ggatactttg ctgtgtagat caaacccaaa tctgtcaatg2701 cttgtactgc atcattcaag tcaacctgcc cctgttttgt cagacatgcc agtgtcagac2761 ttggcatggt cccgaactga ttattgagca actctgcatt tttcacatcc caaactctca2821 ccactccatc tctcccagcc cgagcccctt gattaccacc actcattcct atcatattca2881 ggagagctct tctttggtca agttgctgtg agcttaggtt gcccatatag acacctgcac2941 ttaatggcct ttctgttctg atcacctttg actttaactt ctctagatca gcggctaaga3001 ttaataagtc atctgaggtt agagtcccaa ctctcagtat actcttttgt tgagttgatt3061 ttaattcaac aagattgttg accgcttgat ttaggtccct caaccgtttc aaatcattgt3121 catcccttct ctccttgcgc atcaaccgtt gaacattact gacttcggag aagtcaagtc3181 catgtaaaag agcctgggca tctttcacca cctgtagttt gatgttggag cagtaaccag3241 ataattccct cctcaaagat tgtgtccaca aaaaggattt tatttccttt gaggcactca3301 tcgccagatt gttgtgttgt atgcacgcaa caaagaactg agactatctg ccaaaatgac3361 aaaagcaaag cgcaatccaa tagcctagga tccactgtgc g,Lassa virus strain recombinant Josiah segment S, complete sequenceGenBank: HQ688673.1), one or both of the open reading frames thereof, ora fragment or fragments or variants thereof encoding a functionalglycoprotein. Variants can have at least 70%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or more sequence identity to SEQ ID NO:1, or to thesequence encoding an open reading frame thereof.

In some embodiments, the chimeric viral genome includes a nucleic acidsequence encoding the polypeptide

(SEQ ID NO: 2 MGQIVTETQEVPHVIEEVMNIVLIALSVLAVLKOLYNFATCGLVGLVTFLLLCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQYEAMSCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYISRRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHDEEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRDIMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQADNMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIVGKSCPKPHRLN HMGICSCGLYKQPGVPVKWKR,GenBank: HQ688673.1), the polypeptide

(SEQ ID NO: 3 MSASKEIKSFLWTQSLRRELSGYCSNIKLQVVKDAQALLHGLDFSEVSNVQRLMRKERRDDNDLKRLRDLNQAVNNLVELKSTQQKSILRVGTLTSDDLLILAADLEKLKSKVIRTERPLSAGVYMGNLSSQQLDQRRALLNMIGMSGGNQGARAGRDGVVRVWDVKNAELLNNQFGTMPSLTLACLTKQGQVDLNDAVQALTDLGLIYTAKYPNTSDLDRLTQSHPILNMIDTKKSSLNISGYNFSLGAAVKAGACMLDGGNMLETIKVSPQTMDCILKSILKVKKALGMFISDTPGERNPYENILYKICLSGDGWPYTASRTSITGRAWENTVVDLESDGKPQKADSNNSSKSLQSAGFTAGLTYSQLMTLKDAMLQLDPNAKTWMDIEGRPEDPVEIALYQPSSGCYTIHFFREPTDLKQFKQDAKYSHGIDVTDLFATQPGLTSAVIDALPRNMVITCQGSDDIRKLLESQGRKDIKLIDIALSKTDSRKYENAVWDQYKDLCHNHTGVVVEKKKRGGKEEITPHCALMDCIMFDAAVSGGLNTSVLRAVLPRDMVFRTS TPRVVL,GenBank: HQ688673.1), a combination thereof, or a one or more functionalfragments or variants thereof. Variants can have at least 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:2or 3.

b. Ebola G Proteins

In another preferred embodiment, the G protein of VSV is substitutedwith a glycoprotein from an Ebola virus. Ebola virus, along with Marburgvirus, constitutes the family Filoviridae in the order ofMononegavirales (reviewed in Feldmann and Geisbert, Lancet, 377(9768):849-862 (2011), and Sanchez, et al., Filoviridae: Marburg and Ebolaviruses. In: Knipe, D M.; Howley, P M., editors. Fields virology.Philadelphia: Lippincott Williams & Wilkins; 2006. p. 1409-1448).Filoviruses are enveloped, non-segmented, negative-stranded RNA viruseswith filamentous particles. Ebola virus particles have a uniformdiameter of 80 nm but can greatly vary in length, with lengths up to14000 nm. The genome includes seven genes in the order 3′ leader,nucleoprotein, virion protein (VP) 35, VP40, glycoprotein, VP30, VP24,RNA-dependent RNA polymerase (L)-5′ trailer. With the exception of theglycoprotein gene, all genes are monocistronic, encoding for onestructural protein. The inner ribonucleoprotein complex of virionparticles consists of the RNA genome encapsulated by the nucleoprotein,which associates with VP35, VP30, and RNA-dependent RNA polymerase toform the functional transcriptase-replicase complex. Additionally, theproteins of the ribonucleoprotein complex have other functions, forexample, VP35 is an antagonist of interferon; VP40 is a matrix proteinand modulates particle formation; VP24, is structural,membrane-associated protein that also interferes with interferonsignaling.

The glycoprotein is the only transmembrane surface protein of the virusand forms trimeric spikes consisting of glycoprotein 1 and glycoprotein2—two di-sulphide linked furin-cleavage fragments (Sanchez, et al.,Filoviridae: Marburg and Ebola viruses. In: Knipe, D M.; Howley, P M.,editors. Fields virology. Philadelphia: Lippincott Williams & Wilkins;2006. p. 1409-1448). The primary product of the GP gene is a solubleglycoprotein (sGP) that is also secreted from infected cells, acharacteristic distinguishing it from other Mononegavirales (Sanchez, etal., Proc Natl Acad Sci USA, 93:3602-3607 (1996), Volchkov, et al.,Virology, 214:421-430 (1995)). Nucleic acid sequences encoding Ebolaglycoprotein, the mechanism of transcription/translation yieldingfunctional Ebola glycoprotein, Ebola glycoprotein amino acid sequences,and the structure and function of Ebola glycoprotein are well known inthe art and discussed in, for example, Lee and Saphire, Future Virology,4(6):621-635 (2009), Sanchez, Proc Natl Acad Sci USA., 93(8):3602-3607(1996), Volchkov, et al., Virology, 214(2):421-430 (1995), Gire et al,Science, 345: 1369-1372 (2014)).

The Ebola virus glycoprotein can be from a naturally occurring virus, ora virus modified, for example, to increase or decrease the virulence ofthe virus, and/or increase the specificity or infectivity of the viruscompared to the parental strain or serotype. Suitable species of Ebolavirus from which the glycoprotein of the chimeric VSV virus can bederived are known in the art and include, for example, Sudan ebolavirus(SEBOV), Zaire ebolavirus (ZEBOV), Côte d'Ivoire ebolavirus (also knownand here referred to as Ivory Coast ebolavirus (ICEBOV)), Restonebolavirus (REBOV), and Bundigbugyo ebolavirus (BEBOV) (Geibert andFeldmann, J. Infect. Dis., 204 (suppl 3): S1075-S1081 (2011)).Preferably, the chimeric virus's genome, or plasmid(s) encoding thevirus's genome encode the entire Ebola virus glycoprotein (GP), suchthat the glycoprotein is expressed and contributes to formation of thechimeric virus's envelope. In some embodiments, the chimeric virus'sgenome, or plasmid(s) encoding the virus's genome encode less than theentire Ebola virus glycoprotein. For example, in some embodiments, theviral genome or plasmid(s) encoding recombinant viral genome encodes aglycoprotein that is a truncated or variant GP. In some embodiments, thechimeric virus's genome, or plasmid(s) encoding the virus's genomeencode full length, truncated, or variant GP1, GP2, or a combinationthereof.

In some embodiment, the chimeric viral genome includes the nucleic acidsequence

(SEQ ID NO: 6)   1 atgggcgtta caggaatatt gcagttacct cgtgatcgat tcaagaggac atcattcttt  61 ctttgggtaa ttatcctttt ccaaagaaca ttttccatcc cacttggagt catccacaat 121 agcacattac aggttagtga tgtcgacaaa ctagtttgtc gtgacaaact gtcatccaca 181 aatcaattga gatcagttgg actgaatctc gaagggaatg gagtggcaac tgacgtgcca 241 tctgcaacta aaagatgggg cttcaggtcc ggtgtcccac caaaggtggt caattatgaa 301 gctggtgaat gggctgaaaa ctgctacaat cttgaaatca aaaaacctga cgggagtgag 361 tgtctaccag cagcgccaga cgggattcgg ggcttccccc ggtgccggta tgtgcacaaa 421 gtatcaggaa cgggaccgtg tgccggagac tttgccttcc ataaagaggg tgctttcttc 481 ctgtatgatc gacttgcttc cacagttatc taccgaggaa cgactttcgc tgaaggtgtc 541 gttgcatttc tgatactgcc ccaagctaag aaggacttct tcagctcaca ccccttgaga 601 gagccggtca atgcaacgga ggacccgtct agtggctact attctaccac aattagatat 661 caggctaccg gttttggaac caatgagaca gagtacttgt tcgaggttga caatttgacc 721 tacgtccaac ttgaatcaag attcacacca cagtttctgc tccagctgaa tgagacaata 781 tatacaagtg ggaaaagyag caataccacg ggaaaactaa tttggaaggt caaccccgaa 841 attgatacaa caatcgggga gtgggccttc tgggaaacta aaaaaaacct cactagaaaa 901 attcgcagtg aagagttgtc tttcacagtt gtatcaaacg gagccaaaaa catcagtggt 961 cagagtccgg cgcgaacttc ttccgaccca gggaccaaca caacaactga agaccacaaa1021 atcatggctt cagaaaattc ctctgcaatg gttcaagtgc acagtcaagg aagggaagct1081 gcagtgtcgc atctaacaac ccttgccaca atctccacga gtccccaatc cctcacaacc1141 aaaccaggtc cggacaacag cacccataat acacccgtgt ataaacttga catctctgag1201 gcaactcaag ttgaacaaca tcaccgcaga acagacaacg acagcacagc ctccgacact1261 ccctctgcca cgaccgcagc cggaccccca aaagcagaga acaccaacac gagcaagagc1321 actgacttcc tggaccccgc caccacaaca agtccccaaa accacagcga gaccgctggc1381 aacaacaaca ctcatcacca agataccgga gaagagagtg ccagcagcgg gaagctaggc1441 ttaattacca atactattgc tggagtcgca ggactgatca caggcgggag aagaactcga1501 agagaagcaa ttgtcaatgc tcaacccaaa tgcaacccta atttacatta ctggactact1561 caggatgaag gtgctgcaat cggactggcc tggataccat atttcgggcc agcagccgag1621 ggaatttaca tagaggggct aatgcacaat caagatggtt taatctgtgg gttgagacag1681 ctggccaacg agacgactca agctcttcaa ctgttcctga gagccacaac tgagctacgc1741 accttttcaa tcctcaaccg taaggcaatt gatttcttgc tgcagcgatg gggcggcaca1801 tgccacattc tgggaccgga ctgctgtatc gaaccacatg attggaccaa gaacataaca1861 gacaaaattg atcagattat tcatgatttt gttgataaaa cccttccgga ccagggggac1921 aatgacaatt ggtggacagg atggagacaa tggataccgg caggtattgg agttacaggc1981 gttataattg cagttatcgc tttattctgt atatgcaaat ttgtctttta g.

SEQ ID NO:6 a nucleic acid encoding a full-length (non-secreted)glycoprotein gene found in GenBank accession NC_002549 nt 6039-8068.Note that the GenBank sequence over this region is 2030 nt long, whereasSEQ ID NO:6 is 2031 nt in length. This difference derives from the factthat the GenBank sequence is based on the genomic RNA sequence, whereasthe sequence below is based on the mRNA sequence that has been ‘edited’by the viral polymerase to include an extra ‘A’ nucleotide between6918-6924 of the GenBank sequence. Therefore, in some embodiments, thechimeric viral genome includes a nucleic acid encoding SEQ ID NO:6, orthe variant thereof encoded by GenBank accession NC_002549 nt 6039-8068,or a fragment or variant thereof encoding a functional glycoprotein.Variants can have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or more sequence identity to SEQ ID NO:6, or to the sequence encoding anopen reading frame thereof.

In some embodiments, the chimeric viral genome includes a nucleic acidsequence encoding the polypeptide

(SEQ ID NO: 7) MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSTPLGVIHNSTLQVSDVDKINCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYAGTTFAEGVVAFLILPQAKKDFFSSEPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPULLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAYNISGQSPARTSSBPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPOLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGTTKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREATVNAUKONPNLHYWTTQDEGAAIGLAWIFYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSTLINIRKAIDFLWRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLITOGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKF VF,or a functional fragment or variant thereof. Variants can have at least70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO:7.

In some embodiments, the chimeric viral genome includes the nucleic acidsequence

(SEQ ID NO: 8)   1 atgggcgtta caggaatatt gcagttacct cgtgatcgat tcaagaggac atcattcttt  61 ctttgggtaa ttatcctttt ccaaagaaca ttttccatcc cacttggagt catccacagt 121 agcacattac aggttagtga tgtcgacaaa ctagtttgtc gtgacaaact gtcatccaca 181 aatcaattga gatcagttgg actgaatctc gaagggaatg gagtggcaac tgacgtgcca 241 tctgcaacta aaagatgggg cttcaggtcc ggtgtcccac caaaggtggt caattatgaa 301 gctggtgaat gggctgaaaa ctgctacaat cttgaaatca aaaaacctga cgggagtgag 361 tgtctaccag cagcgccaga cgggattcgg ggettocccc ggtgccggta tgtgcacaaa 421 gtatcaggaa cgggaccgtg tgccggagac tttgccttcc ataaagaggg tgctttcttc 481 ctgtatgatc gacttgcttc cacagttatc taccgaggaa cgactttcgc tgaaggtgtc 541 gttgcatttc tgatactgcc ccaagctaag aaggacttct tcagctcaca ccccttgaga 601 gagccggtca atgcaacgga ggacccgtct agtggctact attctaccac aattagatat 661 caggctaccg gttttggaac caatgagaca gagtacttgt tcgaggttga caatttgacc 721 tacgtccaac ttgaatcaag attcacacca cagtttctgc tccagctgaa tgagacaata 781 tatacaagtg ggaaaaggag caataccacg ggaaaactaa tttggaaggt caaccccgaa 841 attgatacaa caatcgggga gtgggccttc tgggaaacta aaaaaaacct cactagaaaa 901 attcgcagtg aagagttgtc tttctctaga gcaggactga tcacaggcgg gagaagaact 961 cgaagagaag caattgtcaa tgctcaaccc aaatgcaacc ctaatttaca ttactggact1021 actcaggatg aaggtgctgc aatcggactg gcctggatac catatttcgg gccagcagcc1081 gagggaattt acatagaggg gctaatgcac aatcaagatg gtttaatctg tgggttgaga1141 cagctggcca acgagacgac tcaagctctt caactgttcc tgagagccac aactgagcta1201 cgcacctttt caatcctcaa ccgtaaggca attgatttct tgctgcagcg atggggcggc1261 acatgccaca ttctgggacc ggactgctgt atcgaaccac atgattggac caagaacata1321 acagacaaaa ttgatcagat tattcatgat tttgttgata aaacccttcc ggaccagggg1381 gacaatgaca attggtggac aggatggaga caatggatac cggcaggtat tggagttaca1441 ggcgttgtaa ttgcagttat cgctttattc tgtatatgca aatttgtctt ttag,or a fragment or variant thereof encoding a functional glycoprotein.Variants can have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or more sequence identity to SEQ ID NO:8.

In some embodiments, the chimeric viral genome includes a nucleic acidsequence encoding the polypeptide

(SEQ ID NO: 9) MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIESSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGICAGDFAFBEEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKAFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYIFENDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPFIATTIGEWAFWETKKNLTRKIRSEELSFSRAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTIEGLMHNQDGLICGLRQLANETWALQLELRATTELRTFSILNRKAIDFLLQRWGGTCHILGPECCIEPEDWTKNITDKIDQIIHDFVDKTIPDQGDNDNWWTGWRQWIPAGIGVT GVVIAVIALFCICKFVF,or a functional fragment or variant thereof. Variants can have at least70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO:9.

In some embodiments, the Ebola G protein is lacking the mucin domain,(e.g., about amino acids 309-489 of a full length G protein), and/or hasone or more substitutions (e.g. as described in Wong, et al., J Virol.,84(1):163-75 (2010), and in the examples below).

3. Additional Transgenes

Viruses can be modified to express one or more additional transgenes,separately or as a part of other expressed proteins. The viral genome ofVSV has the capacity to accommodate additional genetic material. Atleast two additional transcription units, totaling 4.5 kb, can be addedto the genome, and methods for doing so are known in the art. The addedgenes are stably maintained in the genome upon repeated passage(Schnell, et al., EMBO Journal, 17:1289-1296 (1998); Schnell, et al.,PNAS, 93: 11359-11365 (1996); Schnell, et al., Journal of Virology,70:2318-2323 (1996); Kahn, et al., Virology, 254, 81-91 (1999)).

In preferred embodiments the viruses are modified to include a geneencoding a therapeutic protein, an antigen, a detectable marker orreporter, a targeting moiety, or a combination thereof. In someembodiments, the gene is placed in the first gene position in the VSVbackground. Given the nature of VSV protein expression, genes in thefirst position generate the highest expression of any gene in the virus,with a 3′ to 5′ decrease in gene expression. The chimeric VSV can alsobe constructed to contain two different and independent genes placed inthe first and second gene position of VSV. For example, van den Pol andDavis, et al., J. Virol., 87(2):1019-1034 (2013), describes thegeneration of a highly attenuated VSV virus by adding two (reporter)genes to the 3′ end of the VSV genome, thereby shifting the NPMGL genesfrom positions 1 to 5 to positions 3 to 7. This strategy can be used toallow strong expression of genes coding for any combination of twoheterologous proteins, for example two therapeutic proteins, atherapeutic protein and reporter, or an immunogenic protein and areporter that could be useful to track the virus in a clinicalsituation.

a. Therapeutic Proteins and Reporters

Chimeric VSV viruses can be engineered to include one or more additionalgenes that encode a therapeutic protein or a reporter. Suitabletherapeutic proteins, such as cytokines or chemokines, are known in theart, and can be selected depending on the use or disease to be treated.Preferred cytokines include, but are not limited to, granulocytemacrophage colony stimulating factor (GM-CSF), tumor necrosis factoralpha (TNFα), tumor necrosis factor beta (TNFβ), macrophage colonystimulating factor (M-CSF), interleukin-1 (IL-1), interleukin-2 (IL-2),interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15),interleukin-21 (IL-21), interferon alpha (IFNα), interferon beta (IFNβ),interferon gamma (IFNγ), and IGIF, and variants and fragments thereof.

Suitable chemokines include, but are not limited to, an alpha-chemokineor a beta-chemokine, including, but not limited to, a C5a, interleukin-8(IL-8), monocyte chemotactic protein I alpha (MIP1α), monocytechemotactic protein 1 beta (MIP1β), monocyte chemo-attractant protein 1(MCP-1), monocyte chemo-attractant protein 3 (MCP-3), plateletactivating factor (PAFR), N-formyl-methionyl-leucyl-[³H]phenylalanine(FMLPR), leukotriene B₄, gastrin releasing peptide (GRP), RANTES,eotaxin, lymphotactin, IP10, 1-309, ENA78, GCP-2, NAP-2 and MGSA/gro,and variants and fragments thereof.

Particularly preferred genes include those that encode proteins thatup-regulate an immune attack on infected tumors such as IL-28, IL-2,FLT3L, and GM-CSF (Ali, et al., Cancer Res, 65:7194-7204 (2005); Barzon,et al., Methods Mol. Biol., 542:529-549 (2009); Wongthida, et al., Hum.Gene Ther., 22:1343-53 (2011). Other therapeutic proteins that have beensuccessfully engineered into VSV or other viruses include IL2, IL-4,IL-7, IL-12, and TRAIL (Jinush, et al., Cancer Science, 100, 1389-1396.(2009)). The virus can also be engineered to include one or more genesencoding a reporter. The reporter can serve as a measure or monitor ofin vivo viral activity. Exemplary reporters are known in the art andinclude, but are not limited to, carcinoembryonic antigen, secretedalkaline phosphatase, and the beta subunit of chorionic gonadotropin.These reporters are released by infected cells into the blood, and canbe measured peripherally to determine viral activity, including viralactivity in the brain (Phuong, et al., Cancer Res., 63:2462-2469 (2003);Peng, et al., Nat. Med., 8:527-531 (2002); Shashkova, et al., CancerGene Ther., 15:61-72 (2008); Hiramatsu, et al., Cancer Science, 100,1389-1396 (2005)).

In some embodiments, the virus's genome is modified to encode adetectable marker or reporter, preferably in the first position. Thedetectable marker allows the user to detect and monitor the location andefficacy of the virus in vivo and in resected tissue ex vivo without theneed for antibodies. Suitable markers are known in the art and include,but are not limited to, LacZ, GFP (or eGFP), and luciferase.

There have been reports of humoral immune response to eGFP and rejectionof eGFP transduced cells following subretinal administration of AAV2 orlentivirus expressing eGFP in animals (Bainbridge, et al., Gene Ther.,10(16):1336-44 (2003), and Doi, K., J. Virol, 78(20): 11327-33 (2004)).Thus, the safety and in vivo persistence of a virus including adetectable marker (e.g., one expressing eGFP) may be different than thatof a virus without the marker, however, these differences can beassessed by one of skill in the art using methods known in the art andthe methods described in the Examples. As discussed in more detailabove, in the particular case of VSV, adding a gene added to the firstposition typically attenuates the virulence of VSV (Wollmann, et al., J.Virol., 84(3):1563-73 (2010)). Therefore, in some embodiments, chimericVSV that include a marker such as GFP in the first position may have animproved safety profile compared viruses without it.

b. Viruses Engineered to Deliver Vaccine Antigens

As discussed in more detail below, the virus can be a vaccine vectorthat serves as an immunogen for eliciting an immune response against adisease. This can be accomplished by cloning an antigen of an unrelateddisease into the chimeric VSV virus. VSV viruses expressing foreignviral glycoproteins have shown promise as a vaccine vectors (Roberts, etal., J. Virol. 73:3723-3732 (1999), Rose, et al., Cell, 106:539-549(2001), Jones, et al., Nat. Med. 11:786-790 (2005)). Additionally,recombinant VSVs are able to accommodate large inserts and multiplegenes in their genomes. This ability to incorporate large gene insertsin replication-competent viruses offers advantages over other RNA or DNAvirus vectors, such as those based on alphaviruses, REO virus,poliovirus, and parvovirus.

VSV viruses can be engineered to incorporate one or more nucleic acidsequences encoding one or more non-native or heterologous immunogenicantigens. One or more native VSV genes may be truncated or deleted tocreate additional space for the sequence encoding the immunogenicantigen. When expressed by the VSV virus administered to a patient inneed thereof, the immunogenic antigen produces prophylactic ortherapeutic immunity against a disease or disorder. Immunogenic antigenscan be expressed as a fusion protein with other polypeptides including,but not limited to, native VSV polypeptides, or as a non-fusion protein.By way of non-limiting examples, the antigen can be a protein orpolypeptide derived from a virus, bacterium, parasite, plant, protozoan,fungus, tissue or transformed cell such as a cancer or leukemic cell.Antigens may be expressed as single antigens or may be provided incombination.

Because the substitution of the Lassa glycoprotein for the VSVglycoprotein generates a chimeric virus that appears far safer than VSVsthat contain the VSV glycoprotein, yet still retains the broad spectrumof cells to which it can bind, the chimeric virus can serve as avaccination platform for a wide variety of microbial pathogens,including but not limited to, HIV, influenza, polio, measles, mumps,chicken pox, hendra, and others. Additionally, the fact that theLassa-VSV chimeric virus is safe even in the brains of SCID mice lackingthe normal T and B cell systemic immunity, as demonstrated in theExamples below, indicates that a vaccine based on chimeric Lassa-VSVwould be safer than the corresponding vaccine based on VSV that retainedits VSV glycoprotein, and therefore the chimeric Lassa-VSV might beuseful in vaccinating people with depressed immune systems, for instancethose with AIDS or those with genetically compromised immune systems, orpatients with attenuated immunity related to ongoing cancer. The targetof the vaccine could either be a type of cancer cell as a cancertreatment. Alternately, the target could be any of a large number ofmicrobial pathogens.

c. Targeting Domains

Viruses can be engineered to include one or more additional genes thattarget the virus to cells of interest, see for example U.S. Pat. No.7,429,481. In preferred embodiments, expression of the gene results inexpression of a ligand on the surface of the virus containing one ormore domains that bind to antigens, ligands or receptors that arespecific to tumor cells, or are up-regulated in tumor cells compared tonormal tissue. Appropriate targeting ligands will depend on the targetcell or cancer of interest and will be known to those skilled in theart. For example, glioma stem cells are reported to express CD133 andnestin. Accordingly, in some embodiments, the viruses are engineered toexpress a targeting moiety that bind to CD133 or nestin.

It is believed that the Lassa glycoprotein is important for targetingchimeric Lassa-VSV virus to cells, and contributes to the desirableoncolytic profile exhibited by the chimeric Lassa-VSV virus.Accordingly, in preferred embodiments, any additional targeting ligandsor moieties engineered into the virus do not reduce and preferablyenhance the oncolytic activity or profile of the virus.

4. Exemplary Chimeric Viruses

Exemplary chimeric VSV viruses with Lassa virus glycoprotein are knownin the art. The viruses can be used in the disclosed methods of use andtreatment with or without one or more modifications, such as thosediscussed above.

An exemplary virus is described in Jae, et al., Science,340(6151):479-483 (2013). Briefly, recombinant VSV expressing eGFP andthe Lassa virus glycoprotein (rVSV-GP-LASV) was cloned and recovered asfollows: the open reading frame encoding LASV-GP (strain Josiah,GenBank:HQ688673.1) was amplified using the following primer sequences:5′-GCGACGCGTACCATGGGACAAATAGTGACATTCT-3′ (SEQ ID NO:4) and5′-GGCGGCCGCTCATCTCTTCCATTTCACAGG-3′ (SEQ ID NO:5). Subsequently, thePCR product was sequenced and cloned into the MluI and NotI sites ofpVSVΔG-eGFP-MN (Whelan, et al., Proc. Natl. Acad. Sci. U.S.A.,92(18):8388-92 (1995), and Wong, et al., J. Virol., 84(1):163-75 (2010))thereby replacing the native VSV glycoprotein G coding sequence.Recombinant virus was recovered and amplified as described (Whelan, etal., supra).

The genome of this Lassa-VSV includes an open reading frame encoding aGFP reporter in the first position. This allows easy detection of whichcells are infected, as they turn green. In addition, having a gene addedto the first position attenuates the virulence of VSV (Wollmann, et al.,J. Viral, 84(3):1563-73 (2010)). Accordingly, a chimeric VSV with areporter or other heterologous gene at the first position may beattenuated or less virulent compared to the same virus without areporter or other heterologous gene at the first position. However, asdiscussed in more detail in the Examples below, chimeric Lassa-VSVviruses are both efficacious and safe with or without a reporter, oranother heterologous gene, in the first position. Therefore, theheterologous gene in the first position is optional.

The construction of a recombinant VSV expressing eGFP and the Ebolavirus glycoprotein (rVSV-GP-EBOV) was also described in Wong, et al., J.Virol., 84(1):163-75 (2010).

A second exemplary virus is discussed in Garbutt, et al., J. Virol.,78(10): 5458-5465 (2004) and Geisbert, et al., PLOS, 2(6):537-545(2005), which describe the construction of chimeric VSV viruses having aVSV, Indiana serotype background and a glycoprotein from Lassa virus,strain Josiah. A plasmid expressing the positive-strand RNA complementof the VSV genome with a site for foreign gene expression is describedin Schnell, et al., J. Virol., 70:2318-2323 (1996). This plasmid(VSVXN2) contains the five VSV genes (nucleoprotein N, phosphoprotein P,matrixprotein M, glycoprotein G, and polymerase L) in order, flanked bythe bacteriophage T7 promoter, the VSV leader, and the hepatitis deltavirus ribozyme, and the T7 terminator sequence. Between the G and the Lgenes, a linker site (XhoI-NheI) is present, flanked by atranscriptional start and stop signal for the additional gene to beexpressed. As discussed in Garbutt, et al., J. Virol., 78(10): 5458-5465(2004), the plasmid can be modified to delete the G gene, and the openreading frame encoding the transmembrane glycoprotein of Lassa virus(GPC) can be prepared, for example by PCR, and cloned into the XhoI andNheI sites of the modified vector where the G gene has been deleted.

Following cloning, competent cells, for example, BSR-T7 cells, can beco-transfected with the vector and support plasmids encoding the viralribonucleoprotein constituents (e.g., pBS-VSV N, pBS-VSV P, pBS-VSV L)to generate recombinant infectious virus that can be recovered from thesupernatant of the cultured cells. Rescued rVSV can be passaged, onVeroE6 cells, for example, to obtain a virus stock.

VSV-LCMV viruses are described in U.S. Patent Application No.2014/0301992, and 2011/0250188, and U.S. Pat. No. 6,440,730

Additional methods of making and recovering chimeric VSV virus byexpressing full-length cDNA from plasmid(s) are known in the art anddiscussed in more detail below.

B. Pharmaceutical Compositions

Immunizing and therapeutic viruses are typically administered to apatient in need thereof in a pharmaceutical composition. Pharmaceuticalcompositions containing virus may be for systemic or localadministration, such as intratumoral. Dosage forms for administration byparenteral (intramuscular (IM), intraperitoneal (IP), intravenous (IV),intra-arterial, intrathecal or subcutaneous injection (SC)), ortransmucosal (nasal, vaginal, pulmonary, or rectal) routes ofadministration can be formulated. In some embodiments, a therapeuticvirus is delivered by local injection, for example intracranialinjection preferably at or near the tumor site. In a particularembodiment a therapeutic virus is injected directly into the tumor. Thecompositions can be formulated for and delivered via catheter into thetumor resection cavity through convection-enhanced delivery (CED). Insome embodiments an immunizing virus is delivered peripherally,intranasally or by intramuscular injection.

As discussed in more detail below, the virus can also be used as animmunizing virus. The immunizing virus can be the same as a therapeuticvirus but administered prior to a therapeutic administration so that thesubject's immune system is primed to eliminate the virus following thetherapeutic administration. Alternatively, the immunizing virus can bemodified as discussed above to carry a disease antigen and used as partof a vaccine protocol. Immunizing viruses can be delivered peripherally,for example, by the intranasal route or by intramuscular injection.

1. Effective Amounts

As generally used herein, an “effective amount” is that amount which isable to induce a desired result in a treated subject. The desiredresults will depend on the disease or condition to be treated. Theprecise dosage will vary according to a variety of factors such assubject-dependent variables (e.g., age, immune system health, etc.), thedisease, and the treatment being effected. For example, an effectiveamount of immunizing virus generally results in production of antibodyand/or activated T cells against an antigen, or that kill or limitproliferation of or infection by a pathogen. An effective amount of theimmunizing virus can be an amount sufficient to reduce neurovirulence ofthe therapeutic virus compared to administration of the therapeuticvirus without first administering the immunizing virus.

Therapeutically effective amounts of the therapeutic viruses disclosedherein used in the treatment of cancer will generally kill tumor cellsor inhibit proliferation or metastasis of the tumor cells. Symptoms ofcancer may be physical, such as tumor burden, or biological such asproliferation of cancer cells. The actual effective amounts of virus canvary according to factors including the specific virus administered, theparticular composition formulated, the mode of administration, and theage, weight, condition of the subject being treated, as well as theroute of administration and the disease or disorder.

An effective amount of the virus can be compared to a control. Suitablecontrols are known in the art. A typical control is a comparison of acondition or symptom of a subject prior to and after administration ofthe virus. The condition or symptom can be a biochemical, molecular,physiological, or pathological readout. In another embodiment, thecontrol is a matched subject that is administered a differenttherapeutic agent. Accordingly, the compositions disclosed here can becompared to other art recognized treatments for the disease or conditionto be treated.

For example, the virus can be administered in an amount effective toinfect and kill cancer cells, improve survival of a subject with cancer,or a combination thereof. In a particular embodiment, the cancer isglioblastoma.

One of the advantages of the disclosed viruses is that they show littleor no toxicity to normal or healthy cells (e.g., non-cancerous cells).Therefore, in some embodiments the effective amount of virus causeslittle or no destruction of non-cancerous cells. The level ofpathogenicity to normal cells can be compared to the level ofpathogenicity of other VSV oncolytic viruses that do not have G genereplaced with a heterologous G gene. Such viruses are known in the artand include, for example, VSV-1′GFP, VSV-rp30, or VSV-ΔM51.

One important index of oncolytic potential is the ratio of viralreplication in normal/control cells versus tumor or cancer cells. Theseratios serve as an important index of the relative levels of viralreplication in normal and tumor cells. A large ratio indicates greaterreplication in cancer cells than in control cells. In preferredembodiments, the ratio of replication of normal cells:target cells isgreater than about 1:100, preferably greater than about 1:250, morepreferable greater than about 1:500, most preferably greater than about1:1000. In some embodiments, the oncolytic potential of the disclosedviruses is larger than the oncolytic potential of other VSV oncolyticviruses that do not have G gene replaced with a heterologous G gene, forexample, VSV-1′GFP, VSV-rp30, or VSV-AM51.

2. Dosages

Appropriate dosages can be determined by a person skilled in the art,considering the therapeutic context, age, and general health of therecipient. The selected dosage depends upon the desired therapeuticeffect, on the route of administration, and on the duration of thetreatment desired. Active virus can also be measured in terms ofplaque-forming units (PFU). A plaque-forming unit can be defined asareas of cell lysis (CPE) in monolayer cell culture, under overlayconditions, initiated by infection with a single virus particle.Generally dosage levels of virus between 10² and 10¹² PFU areadministered to humans. Virus is typically administered in a liquidsuspension, in a volume ranging between 10 μl and 100 ml depending onthe route of administration. Generally, dosage and volume will be lowerfor intratumoral injection as compared to systemic administration orinfusion. The dose may be administered once or multiple times. Whenadministered locally, virus can be administered to humans at dosagelevels between 10² and 10⁸ PFU. Virus can be administered in a liquidsuspension, in a low volume. For example, the volume for localadministration can range from about 20 nl to about 200 μl. Multipledoses can be administered. In some embodiment, multiple injections areused to achieve a single dose. Systemic or regional administration viasubcutaneous, intramuscular, intra-organ, or intravenous administrationcan have higher volumes, for example, 10 to 100 ml.

3. Formulations

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredients. The term “pharmaceutically-acceptable carrier”means one or more compatible solid or liquid fillers, diluents orencapsulating substances which are suitable for administration to ahuman or other vertebrate animal. The term “carrier” refers to anorganic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application.

Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thecompositions may be administered in combination with one or morephysiologically or pharmaceutically acceptable carriers, thickeningagents, co-solvents, adhesives, antioxidants, buffers, viscosity andabsorption enhancing agents and agents capable of adjusting osmolarityof the formulation. Proper formulation is dependent upon the route ofadministration chosen. If desired, the compositions may also containminor amounts of nontoxic auxiliary substances such as wetting oremulsifying agents, dyes, pH buffering agents, or preservatives. Theformulations should not include membrane disrupting agents which couldkill or inactivate the virus.

a. Formulations for Local or Parenteral Administration

In a preferred embodiment, compositions including oncolytic virusesdisclosed herein, are administered in an aqueous solution, by parenteralinjection. Injection includes, but it not limited to, local,intratumoral, intravenous, intraperitoneal, intramuscular, orsubcutaneous injection. The formulation may also be in the form of asuspension or emulsion. In general, pharmaceutical compositions areprovided including effective amounts of virus, and optionally includepharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents such as sterile water, buffered saline of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; andoptionally, additives such as anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives and bulking substances (e.g., lactose,mannitol). Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Apreferred solution is phosphate buffered saline or sterile saline.

b. Formulations for Mucosal Administration

In some embodiments, the compositions are formulated for mucosaladministration, such as through nasal, pulmonary, or buccal delivery.

Mucosal formulations may include one or more agents for enhancingdelivery through the nasal mucosa. Agents for enhancing mucosal deliveryare known in the art, see, for example, U.S. Patent Application No.200910252672 to Eddington, and U.S. Patent Application No. 2009/0047234to Touitou. Acceptable agents include, but are not limited to, chelatorsof calcium (EDTA), inhibitors of nasal enzymes (boro-leucin, aprotinin),inhibitors of muco-ciliar clearance (preservatives), solubilizers ofnasal membrane (cyclodextrin, fatty acids, surfactants) and formation ofmicelles (surfactants such as bile acids, Laureth 9 andtaurodehydrofusidate (STDHF)). Compositions may include one or moreabsorption enhancers, including surfactants, fatty acids, and chitosanderivatives, which can enhance delivery by modulation of the tightjunctions (TJ) (B. J. Aungst, et al., J. Pharm. Sci. 89(4):429-442(2000)). In general, the optimal absorption enhancer should possess thefollowing qualities: its effect should be reversible, it should providea rapid permeation enhancing effect on the cellular membrane of themucosa, and it should be non-cytotoxic at the effective concentrationlevel and without deleterious and/or irreversible effects on thecellular or virus membrane, Intranasal compositions maybe administeredusing devices known in the art, for example a nebulizer.

III. Methods of Use

A. Methods of Treatment

1. Administration of Therapeutic Virus

The disclosed chimeric VSV viruses can be administered to a subject inneed thereof in an amount effective to treat a disease or disorder, forexample, cancer. Pharmaceutical compositions including a chimeric virusmay be administered once or more than once, for example 2, 3, 4, 5, ormore times. Serial administration of chimeric virus may occur days,weeks, or months apart. As discussed in more detail below, boosters ofimmunizing virus may be administered between therapeutic treatments. Itmay be particularly preferable to administer a booster of immunizingvirus if there are lengthy delays between treatments with therapeuticvirus, for example, one or more years.

Virus can be administered peripherally, or can be injected directly intoa tumor, for example a tumor within the brain. In addition, virus can beused after resection of the main body of the tumor, for example byadministering directly to the remaining adjacent tissue after surgery,or after a period of one to two weeks to allow recovery of local damage.Adding virus after surgical resection would eliminate any remainingtumor cells that the neurosurgeon did not remove. The injections can begiven at one, or multiple locations. It is also believed that virusadministered systemically can target and kill brain cancers.

In some embodiments, it may be desirable to administer the chimericvirus after or in combination with an immunosuppressant. Treatment withan immunosuppressant during administration with a therapeutic virusallows controlled suppression of the subject's immune system duringadministration of the therapeutic virus. This may be desirable, forexample, if the capacity of the oncolytic virus to kill cancer isreduced due to an earlier administration of the immunizing virus.Treatment with the immunosuppressant is typically transient, and occursduring administration of the virus, particularly when the virus is beingused to treat tumors and/or cancer. Following treatment with thechimeric virus, treatment with the immunosuppressant is discontinued andthe patient's immunity returns. The duration of immunosuppressivetreatment will depend on the condition to be treated. Typically theimmunosuppressive treatment will be long enough for the oncolytic virusto kill cancer cells, reduce tumor size, or inhibit tumor progression.

2. Peripheral Administration of Immunizing Virus

One or more peripheral administrations with an immunizing virus canelicit an adaptive immune response that protects the brain frompotential side-effects of oncolytic virus therapy. The term immunizingvirus includes live virus as well as viral subunits, proteins andfragments thereof, antigenic polypeptides, nucleic acids, and expressionvectors containing nucleic acids encoding viral subunits, proteins, orfragments thereof, or antigenic polypeptides which can be useful ineliciting an immune response. For example, if the immunizing virus is aVSV virus, the immunizing virus includes, but is not limited to, liveVSV virus, the N, P, M, G, or L proteins, or combinations thereof.

The immunizing virus may be the same virus, or a different virus thanthe therapeutic virus. The immunizing virus should initiate an adaptiveimmune response that is sufficient to attenuate, reduce, or prevent theneurovirulence of the therapeutic virus. The therapeutic virusadministered after a first administration of immunizing virus shouldhave reduced neurovirulence compared to therapeutic virus administeredwithout a first administration of immunizing virus. In preferredembodiments, the immunizing virus is similar to the therapeutic virus.For example if the therapeutic virus is a VSV, the immunizing virus ispreferably a VSV, or an antigenic protein or nucleic acid componentthereof. In some embodiments the immunizing virus has an attenuatedphenotype compared to the therapeutic virus. As described above,suitable immunizing viruses include wildtype viruses, as well as mutantand variants thereof. In one preferred embodiment, the immunizing virusis a wildtype virus or an antigenic protein or nucleic acid componentthereof, while the therapeutic virus is a mutant, variant, chimericvirus having the same virus background but reduced neurovirulencecompared to wildtype. In some embodiments, therapeutic viruses may beengineered to express therapeutic proteins or targeting molecules.Immunizing viruses may also be engineered to express additionalproteins, but preferably are not. VSV-G/GFP is a suitable immunizingvirus. The nucleotide sequence for VSV-G/GFP is GenBank AccessionFJ478454.

Immunizing viruses are administered sufficiently prior to therapeuticviruses to elicit an adaptive immune response. Immunizing viruses aretypically administered one or more times at least about 5 days,preferably 1 week, more preferably greater than one week beforeadministration of the therapeutic virus. Immunizing viruses can beadministered up to 1, 2, 3, 4, 5, or more weeks before the therapeuticvirus. Immunizing viruses can be administered up to 1, 2, 3, 4, 5, ormore months before the therapeutic virus. Most preferably the immunizingvirus is administered between about ten days and 12 weeks before thetherapeutic virus.

After an initial administration of the immunizing virus, subsequentbooster immunizations can be administered. For example, it may bedesirable to administer the immunizing virus two or more times. A firstadministration of the immunizing virus is typically provided to apatient in need therefore prior to a first administration of thetherapeutic virus. Subsequent administrations of the immunizing virusmay occur before and/or after a first administration of the therapeuticvirus. In preferred embodiments the immunizing virus is administered twoor more times before the first administration of the therapeutic virus.In a non-limiting example, the immunizing virus is first administered onday 1, a booster of immunizing virus is administered six weeks later onabout day 43, and the therapeutic virus is first administered two weekslater on about day 57.

Various factors may be considered when determining the frequency,dosage, duration, and number of administrations of immunizing virus, aswell as the duration between administration of the immunizing virus andfirst administration of therapeutic virus. For example, the subject'sadaptive immune response can be monitored to assess the effectiveness ofthe immunization. Methods of measuring adaptive immune activation areknown in the art and include antibody profiling, serum analysis forchanges in levels of antibodies, cytokines, chemokines, or otherinflammatory molecules, and cell counts and/or cell profiling usingextracellular markers to assess the numbers and types of immune cellssuch as B cells and T cells.

Immunizing virus is most typically delivered to a subject in needthereof by peripheral administration, and not directly or locally to thesite in need of treatment by therapeutic virus. Peripheraladministration includes intravenous, by injection or infusion,intraperitoneal, intramuscular, subcutaneous, and mucosal such asintranasal delivery. In some embodiments, the composition is deliveredsystemically, by injection or infusion into the circulatory system (i.e.intravenous) or an appropriate lymphoid tissue, such as the spleen,lymph nodes or mucosal-associated lymphoid tissue. The injections can begiven at one, or multiple locations. Preferably the immunizing virus isadministered intranasally or by intramuscular injection, most preferablyby intranasal delivery.

Generally immunizing virus is administered to humans at dosage levelsbetween 10² and 10¹² PFU. Virus is typically administered in a liquidsuspension, in a volume ranging between 10 μl and 100 ml depending onthe route of administration.

It may also be desirable to administer the immunizing virus incombination with one or more adjuvants. These can be incorporated into,administered with, or administered separately from, the immunogenizingvirus. Depending on whether or not the individual is a human or ananimal, the adjuvant can be, but is not limited to, one or more of thefollowing: oil emulsions (e.g., Freund's adjuvant); saponinformulations; virosomes and viral-like particles; bacterial andmicrobial derivatives; immunostimulatory oligonucleotides;ADP-ribosylating toxins and detoxified derivatives; alum; BCG;mineral-containing compositions (e.g., mineral salts, such as aluminiumsalts and calcium salts, hydroxides, phosphates, sulfates, etc.);bioadhesives and/or mucoadhesives; microparticles; liposomes;polyoxyethylene ether and polyoxyethylene ester formulations;polyphosphazene; muramyl peptides; imidazoquinolone compounds; andsurface active substances (e.g. lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol).

3. Vaccination

The chimeric viruses can also serve as an immunogen for generating animmune response against other antigens administered with or cloned intovirus. The safety profile of the disclosed Lassa-VSVs make themparticularly attractive for use as part of a vaccine. Other VSVs canlead to adverse consequence in brain, whereas a Lassa-VSV with anotherantigen, for example, an influenza antigen, would be safer, yeteffective.

For example, in some embodiments, the chimeric virus is a vaccinevector. Experiments conducted with the Lassa-VSV including a GFPreporter discussed below, show that the chimeric virus generates astrong immune response against the virus, and also against the GFPreporter. Accordingly, other proteins could be substituted for GFP.These could include proteins from pathogenic microbes unrelated to Lassavirus or VSV; the Lassa-VSV could serve as a safe vaccine platformagainst many different pathogenic microbes. As described above, VSV canbe engineered to express one or more immunogenic antigens. Expression ofthese antigens in a patient in need thereof presents the antigen to theimmune system and provokes an immune response. Vaccines can beadministered prophylactically or therapeutically. Vaccines can also beadministered according to a vaccine schedule. A vaccine schedule is aseries of vaccinations, including the timing of all doses. Many vaccinesrequire multiple doses for maximum effectiveness, either to producesufficient initial immune response or to boost response that fades overtime. Vaccine schedules are known in the art, and are designed toachieve maximum effectiveness. The adaptive immune response can bemonitored using methods known in the art to measure the effectiveness ofthe vaccination protocol.

4. Immunotherapy

Chimeric VSV viruses wherein the G protein is replaced with aheterologous glycoprotein, for example the glycoprotein from Lassavirus, have been shown to be immunogenic and initiate an up-regulationof both humoral and cellular immunity toward the virus (Geisbert, etal., PLoS Med., 2:e183 (2005) and the Examples below. Therefore, methodsof initiating an immune response against the infected tumor aredisclosed. It is believed that the disclosed chimeric viruses will notonly infect and kill cancer cells, but will enhance an attack by thesystemic immune system on the infected cell-type both during and afterthe virus is eliminated. In this way, the virus can be used to induce animmune response against non-infected target cells. In this way,treatment with the disclosed VSV virus may delay, reduce, or preventreoccurrence of the cancer being treated.

In some methods, the chimeric virus is used to infect targets cells, andthe infected target cells or antigens isolated therefrom are used forperipheral immunization of the subject against the target cells, orantigens thereof. For example, target cells against which an immuneresponse is desired are implanted into a subject. The cells are injectedwith virus which kills the cells and leads to an immune response againstantigens of the cells. The cells can be infected with virus before orafter implantation. For example, the cells are infected with virus invitro prior to injection into the subject. In another embodiment, thesubject is immunized with antigen(s) isolated from tumor cells infectedwith virus in vitro.

The target cell can be any cell to which an immune response is desired.For example, the target cells can be cancer cells against which animmune response is desired. The cancer cells can be from an establishedcell line or primary cancer cells isolated from a subject. For example,the target cells can be cancer cells isolated from a subject in a biopsyor during surgery to remove a tumor. As discussed above, the targetcells can be infected in vitro prior to administration to the subject,or the target cells can be inject by local injection of the virus intothe subject at the site of implantation of the target cells. The cellscan be harvested from and administered back to the same subject.Alternatively, the cells can be harvested from one subject andadministered to a different subject. In this way, the virus can be usedto induce an immune response against a cancer or tumor in a subject thathas the cancer or tumor, or prophylactically prime the immune system toattack a future cancer or tumor that the subject does not yet have.Accordingly, the treatment can be therapeutic, prophylactic, or acombination thereof. In a particular embodiment, this strategy isemployed in combination with surgery in which a tumor is removed from asubject. Cells are isolated from the tumor, infected with virus, andimplanted in the subject. In this way, an immune response is inducedagainst any cancer cells that remain in the subject, for example in themargins and other tissue at the site from which the tumor removed, aswell as circulating cancer cells and metastases throughout the bodyincluding those sites distant from the tumor that was removed. Themethod can also reduce, delay, or prevent recurrence of the cancer.

In some embodiments the isolated target cells are irradiated in amounteffective to prevent cell division, but not to kill the cells, to avoidconcerns about in vivo replication of the target cells followingimplantation. Typically, the cells are implanted into the subjectperipherally. For example, the cells can be injected into the subjectsubcutaneously, intramuscularly, intranasally, intravenously,intraperitoneally, or using another suitable method of peripheraladministration, such as those discussed above. In some embodiments, thetumor cells are expanded in culture for one or generations or passagesbetween isolation and implantation in the subject.

It is believed that VSV infection will increase tumor-specific cytotoxiceffector CD8+ T cells, increase CD4+ T cells, increase production oftumor specific antibodies, or a combination thereof. Therefore, in someembodiments, tumor-specific cytotoxic effector CD8+ T cells primed bychimeric VSV infected tumor cells are administered to a subject in needthereof. The T cells can be harvested from a treated subject, andoptionally expanded in culture, or primed and expanded in vitro.

For example, in a particular embodiment, the method is one of adaptive Tcell therapy. Methods of adoptive T cell therapy are known in the artand used in clinical practice. Generally adoptive T cell therapyinvolves the isolation and ex vivo expansion of tumor specific T cellsto achieve greater number of T cells than what could be obtained byvaccination alone. The tumor specific T cells are then infused intopatients with cancer in an attempt to give their immune system theability to overwhelm remaining tumor via T cells which can attack andkill cancer. Several forms of adoptive T cell therapy can be used forcancer treatment including, but not limited to, culturing tumorinfiltrating lymphocytes or TIL; isolating and expanding one particularT cell or clone; and using T cells that have been engineered torecognize and attack tumors. In the disclosed methods, the tumorsinfected with the chimeric VSV, or isolated components thereof, are usedto prime the T cells. In some embodiments, the T cells are takendirectly from the patient's blood after they have received treatment orimmunization with the virus. Methods of priming and activating T cellsin vitro for adaptive T cell cancer therapy are known in the art. See,for example, Wang, et al., Blood, 109(11):4865-4872 (2007) andHervas-Stubbs, et al., J. Immunol., 189(7):3299-310 (2012). The methodscan be used in conjunction with virus infected cancer cells, or antigensisolated therefrom, to prime and activate T cells against the cancer.

Historically, adoptive T cell therapy strategies have largely focused onthe infusion of tumor antigen specific cytotoxic T cells (CTL) which candirectly kill tumor cells. However, CD4+ T helper (Th) cells can also beused. Th can activate antigen-specific effector cells and recruit cellsof the innate immune system such as macrophages and dendritic cells toassist in antigen presentation (APC), and antigen primed Th cells candirectly activate tumor antigen-specific CTL. As a result of activatingAPC, antigen specific Th1 have been implicated as the initiators ofepitope or determinant spreading which is a broadening of immunity toother antigens in the tumor. The ability to elicit epitope spreadingbroadens the immune response to many potential antigens in the tumor andcan lead to more efficient tumor cell kill due to the ability to mount aheterogeneic response. In this way, adoptive T cell therapy can used tostimulate endogenous immunity.

B. Subjects to be Treated

In general, the disclosed chimeric viruses and methods of treatmentthereof are useful in the context of cancer, including tumor therapy,particular brain tumor therapy.

In a mature animal, a balance usually is maintained between cell renewaland cell death in most organs and tissues. The various types of maturecells in the body have a given life span; as these cells die, new cellsare generated by the proliferation and differentiation of various typesof stem cells. Under normal circumstances, the production of new cellsis so regulated that the numbers of any particular type of cell remainconstant. Occasionally, though, cells arise that are no longerresponsive to normal growth-control mechanisms. These cells give rise toclones of cells that can expand to a considerable size, producing atumor or neoplasm. A tumor that is not capable of indefinite growth anddoes not invade the healthy surrounding tissue extensively is benign. Atumor that continues to grow and becomes progressively invasive ismalignant. The term cancer refers specifically to a malignant tumor. Inaddition to uncontrolled growth, malignant tumors exhibit metastasis. Inthis process, small clusters of cancerous cells dislodge from a tumor,invade the blood or lymphatic vessels, and are carried to other tissues,where they continue to proliferate. In this way a primary tumor at onesite can give rise to a secondary tumor at another site.

The compositions and methods described herein are useful for treatingsubjects having benign or malignant tumors by delaying or inhibiting thegrowth of a tumor in a subject, reducing the growth or size of thetumor, inhibiting or reducing metastasis of the tumor, and/or inhibitingor reducing symptoms associated with tumor development or growth. Theexamples below indicate that the viruses and methods disclosed hereinare useful for treating cancer, particular brain tumors, in vivo.

Malignant tumors which may be treated are classified herein according tothe embryonic origin of the tissue from which the tumor is derived.Carcinomas are tumors arising from endodermal or ectodermal tissues suchas skin or the epithelial lining of internal organs and glands. Thedisclosed compositions are particularly effective in treatingcarcinomas. Sarcomas, which arise less frequently, are derived frommesodermal connective tissues such as bone, fat, and cartilage. Theleukemias and lymphomas are malignant tumors of hematopoietic cells ofthe bone marrow. Leukemias proliferate as single cells, whereaslymphomas tend to grow as tumor masses. Malignant tumors may show up atnumerous organs or tissues of the body to establish a cancer.

The types of cancer that can be treated with the provided compositionsand methods include, but are not limited to, cancers such as vascularcancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone,bladder, brain, breast, cervical, colo-rectal, esophageal, kidney,liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, anduterine. In some embodiments, the disclosed compositions are used totreat multiple cancer types concurrently. The compositions can also beused to treat metastases or tumors at multiple locations.

The disclosed methods are particularly useful in treating brain tumors.Brain tumors include all tumors inside the cranium or in the centralspinal canal. They are created by an abnormal and uncontrolled celldivision, normally either in the brain itself (neurons, glial cells(astrocytes, oligodendrocytes, ependymal cells, myelin-producing Schwanncells, lymphatic tissue, blood vessels), in the cranial nerves, in thebrain envelopes (meninges), skull, pituitary and pineal gland, or spreadfrom cancers primarily located in other organs (metastatic tumors).Examples of brain tumors include, but are not limited to,oligodendroglioma, meningioma, supratentorial ependymona, pineal regiontumors, medulloblastoma, cerebellar astrocytoma, infratentorialependymona, brainstem glioma, schwannomas, pituitary tumors,craniopharyngioma, optic glioma, and astrocytoma.

“Primary” brain tumors originate in the brain and “secondary”(metastatic) brain tumors originate from cancer cells that have migratedfrom other parts of the body. Primary brain cancer rarely spreads beyondthe central nervous system, and death results from uncontrolled tumorgrowth within the limited space of the skull. Metastatic brain cancerindicates advanced disease and has a poor prognosis. Primary braintumors can be cancerous or noncancerous. Both types take up space in thebrain and may cause serious symptoms (e.g., vision or hearing loss) andcomplications (e.g., stroke). All cancerous brain tumors are lifethreatening (malignant) because they have an aggressive and invasivenature. A noncancerous primary brain tumor is life threatening when itcompromises vital structures (e.g., an artery). In a particularembodiment, the disclosed compositions and methods are used to treatcancer cells or tumors that have metastasized from outside the brain(e.g., lung, breast, melanoma) and migrated into the brain.

The Examples below illustrate that Lassa-VSV is oncolytic, but alsonon-toxic to health or normal cells, even when administered directly tothe brain. Therefore, the disclosed viruses are particularly useful fortreating brain cancer, cancer that can metastasize to the brains, forexample lung cancer, breast cancer, and skin cancer such as melanoma.

Although the viruses are particularly safe and useful for treatingcancer in the brain, the cancer does not have to be in the brain. It isbelieved that the chimeric virus are also effective for treating othercancer outside the brain, and can thereof be administered systemicallyin or locally outside the brain. In a particular embodiment, a chimericvirus is used to treat a cancer that could, but has not yet metastasizedto the brain. See, for example, Yarde, et al., Cancer Gene Ther., 2013Nov. 1. doi: 10.1038/cgt.2013.63, which describes that intravenouslyadministered VSVs encoding IFN-β have potent activity againstsubcutaneous tumors in the 5TGM1 mouse myeloma model, without attendantneurotoxicity. However, when 5TGM1 tumor cells were seededintravenously, virus-treated mice with advanced myeloma developedclinical signs suggestive of meningoencephalitis, and leading to deathsthat are believed to be associated with viral toxicity. Histologicalanalysis revealed that systemically administered 5TGM1 cells seed to theCNS, forming meningeal tumor deposits, and that VSV infects and destroysthese tumors. Death is presumably a consequence of meningeal damageand/or direct transmission of virus to adjacent neural tissue.

The disclosed chimeric Lassa-VSV viruses have reduced toxicity fornormal and healthy cells including neurons. Therefore, these viruses area safer, less toxic alternative for treating systemic cancers that canpotential traffic virus into the brain and cause neurotoxicity and evendeath.

As shown the examples below, the chimeric Lassa-VSV was safe in thebrains of SCID mice that have a depressed systemic immunity due tosubstantially reduced T and B cells. Thus, Lassa-VSV should be far saferthan VSV with its normal VSV glycoprotein. This may enable Lassa-VSV tobe used in patients showing depressed immunity, typical of many cancerpatients, and also of patients with AIDS, or with genetic immunedepression. The enhanced safety in the brain may also be of benefit inpatients with compromised blood brain barriers where Lassa-VSV would besafer than VSV in both cancer treatment, and for vaccination againsteither a cancer cell type, or against unrelated (e.g., non-Lassa,non-VSV) pathogenic microbes.

The Examples below also show that the chimeric VSV are effective atinfecting and replicating in colon, prostate, breast, bone, and bladdercancer cells, indicating its oncolytic potential was not restricted toglioma and melanoma brain tumors. It is believed that the disclosedviruses are effective for treating both primary and secondary braintumors, but as peripheral (non-brain) cancers and tumors.

C. Combination Therapies

Administration of the disclosed compositions containing oncolyticviruses may be coupled with surgical, radiologic, other therapeuticapproaches to treatment of tumors and cancers.

1. Surgery

The disclosed compositions and methods can be used as an adjunct tosurgery. Surgery is a common treatment for many types of benign andmalignant tumors. As it is often not possible to remove all the tumorcells from during surgery, the disclosed compositions containingoncolytic virus are particularly useful subsequent to resection of theprimary tumor mass, and would be able to infect and destroy evendispersed tumor cells.

In a preferred embodiment, the disclosed compositions and methods areused as an adjunct or alternative to neurosurgery. The compositions areparticularly well suited to treat areas of the brain that is difficultto treat surgically, for instance high grade tumors of the brain stem,motor cortex, basal ganglia, or internal capsule. High grade gliomas inthese locations are generally considered inoperable. An additionalsituation where an oncolytic virus may be helpful is in regions wherethe tumor is either wrapped around critical vasculature, or in an areathat is difficult to treat surgically.

2. Therapeutic Agents

The viral compositions can be administered to a subject in need thereofalone or in combination with one or more additional therapeutic agentsselected based on the condition, disorder or disease to be treated. Adescription of the various classes of suitable pharmacological agentsand drugs may be found in Goodman and Gilman, The Pharmacological Basisof Therapeutics, (11th Ed., McGraw-Hill Publishing Co.) (2005).

Additional therapeutic agents include conventional cancer therapeuticssuch as chemotherapeutic agents, cytokines, chemokines, and radiationtherapy. The majority of chemotherapeutic drugs can be divided into:alkylating agents, antimetabolites, anthracyclines, plant alkaloids,topoisomerase inhibitors, and other antitumour agents. All of thesedrugs affect cell division or DNA synthesis and function in some way.Additional therapeutics include monoclonal antibodies and the tyrosinekinase inhibitors e.g., imatinib mesylate (GLEEVEC® or GLIVEC®), whichdirectly targets a molecular abnormality in certain types of cancer(chronic myelogenous leukemia, gastrointestinal stromal tumors).

Representative chemotherapeutic agents include, but are not limited to,amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,docetaxel, doxorubicin, epipodophyllotoxins, epirubicin, etoposide,etoposide phosphate, fludarabine, fluorouracil, gemcitabine,hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin,liposomal doxorubicin, liposomal daunorubicin, lomustine,mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate,mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin,teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa,tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine,vinorelbine, taxol and derivatives thereof, trastuzumab (HERCEPTIN®),cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab(AVASTIN®), and combinations thereof. Representative pro-apoptoticagents include, but are not limited to, fludarabinetaurosporine,cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), andcombinations thereof.

Preferred chemotherapeutics will affect tumors or cancer cells, withoutdiminishing the activity of the virus. For example, in a preferredembodiment, the additional therapeutic agent inhibits proliferation ofcancer cells without affecting targeting, infectivity, or replication ofthe virus.

a. Anticancer Agents

The compositions can be administered with an antibody or antigen bindingfragment thereof specific for growth factor receptors or tumor specificantigens. Representative growth factors receptors include, but are notlimited to, epidermal growth factor receptor (EGFR; HER1); c-erbB2(HER2); c-erbB3 (HER3); c-erbB4 (HER4); insulin receptor; insulin-likegrowth factor receptor 1 (IGF-1R); insulin-like growth factor receptor2/Mannose-6-phosphate receptor (IGF-II RIM-6-P receptor); insulinreceptor related kinase (IRRK); platelet-derived growth factor receptor(PDGFR); colony-stimulating factor-1receptor (CSF-1R) (c-Fms); steelreceptor (c-Kit); Flk2/Flt3; fibroblast growth factor receptor 1(Flg/Cek1); fibroblast growth factor receptor 2 (Bek/Cek3/K-Sam);Fibroblast growth factor receptor 3; Fibroblast growth factor receptor4; nerve growth factor receptor (NGFR) (TrkA); BDNF receptor (TrkB);NT-3-receptor (TrkC); vascular endothelial growth factor receptor 1(Flt1); vascular endothelial growth factor receptor 2/Flk1/KDR;hepatocyte growth factor receptor (HGF-R/Met); Eph; Eck; Eek;Cek4/Mek4/HEK; Cek5; Elk/Cek6; Cek7; Sek/Cek8; Cek9; Cek10; HEK11; 9Ror1; Ror2; Ret; Axl; RYK; DDR; and Tie.

b. Therapeutic Proteins

It may be desirable to administer the disclosed compositions incombination with therapeutic proteins. VSV is an effective oncolyticvirus, in-part, by taking advantage of defects in the interferon system.Administration of therapeutic proteins such as IFN-α, or IFN-α/β pathwayinducer polyriboinosinic polyribocytidylic acid [poly(I:C)] areeffective in protecting normal cells from the oncolytic activity, whileleaving the tumor cells susceptible to infection and death (Wollmann, etal. J. Virol., 81(3): 1479-1491 (2007). Therefore, in some embodiments,the disclosed compositions are administered in combination with atherapeutic protein to, reduce infectivity and death of normal cells.

Other therapeutic proteins that can be administered in combination withthe disclosed viruses include those provided above as therapeuticproteins that can be engineered into the virus. Accordingly, thetherapeutic virus can be part of the virus itself, or administeredseparately. In some embodiments, the virus includes one or moretherapeutic proteins and one more therapeutic proteins are administeredseparately.

c. Immuno-Suppressants

As discussed throughout and demonstrated in the Examples below, thedisclosed chimeric VSV viruses generally, show a reduced probability ofinfecting normal brain cells, but still have a good oncolytic capacity.One limitation of oncolytic viruses in general is that the adaptiveimmune system can up-regulate its antiviral response and eliminate thevirus before the virus has had a chance to maximally infect tumor cells.Although it is important for the adaptive immune system to eliminate thechimeric VSV from the subject, the virus should remain in the subjectlong enough to infect and kill as many tumor cells as possible balancedagainst the pathogenicity of the virus to normal cells of the subject.Temporary concomitant immune-suppression has been identified as astrategy to enhance the efficacy of other oncolytic viruses (HSV,adenovirus, vaccinia) that are human pathogens and face pre-existingimmunity (Fukuhara, et al., Curr. Cancer Drug Targets, 7:149-155 (2007);Lun, et al., Clin. Cancer Res., 15:2777-2788 (2009)). Therefore, thevirus can be administered to the subject in combination with temporaryconcomitant immune suppression.

In some embodiments, the virus is administered in combination with anagent that reduces or attenuates the intrinsic IFN-mediated immuneresponses that can eliminate the virus before it has achieved maximaltumor destruction. In preferred embodiments, the attenuation of theintrinsic IFN-mediated immune responses enhances the rate of recombinantVSV-mediated tumor destruction without increasing infection of normalcells. This strategy should also reduce the initiation of the adaptiveimmune response which is enhanced by the innate immune response, givingthe virus more time to complete its oncolytic actions.

Paglino, et al., J. Virol., 85:9346-58 (2011) showed that a cancer cellhighly resistant to VSV could be infected by blocking the IFN responseto VSV with one of three IFN blockers, valproate, the vacccinia proteinB18R, or Jak inhibitor 1. Valproate crosses the blood brain barrier asevident in its use to treat epilepsy. It is already approved forclinical use in humans (for attenuating epilepsy), and like many otherhistone deacetylase (HDAC) inhibitors, it has an intrinsic anti-tumorproperty, independent of oncolytic virus infection, that reduces gliomaand other tumor growth in the brain (Chateauvieux, et al., J. Biomed.Biotechnol., 479364. Epub 2010 July 29 (2010); Fu, et al., Neuro.Oncol., 12:328-340 (2010); Su, et al., Clin. Cancer Res., 17:589-597(2011). Similarly, the HDAC inhibitor vorinostat (ZOLINZA®) is approvedby the FDA for the treatment of cutaneous T-cell lymphoma (Glaser K B,Biochem. Pharmacal., 74:659-671 (2007)). Vorinostat on its own appearsto penetrate brain tumors and to increase survival of patients withglioblastoma, and animal studies have shown that valproate can increaseinfection by viruses in tumors with minimal increased collateral damage.Valproate increased survival substantially in tumor bearing animalstreated with HSV (Otsuki, et al., Mol. Ther., 16:1546-1555 (2008)). Inone particular ease study a pediatric anaplastic astrocytoma that wasresistant to chemotherapy and irradiation, underwent a substantialregression after combined treatment with oral valproate and oncolyticattenuated Newcastle disease virus Wagner, et al., APMIS, 114:731-743(2006)).

Other HDAC inhibitors have been shown to enhance viral cancer celltargeting and viral replication by vaccinia (MacTavish, et al., PLoSOne, 5:e14462 (2010) and VSV (Nguyen, et al., Proc. Natl. Acad. Sci.,USA 105:14981-14986 (2008)) without substantially altering infection innormal non-cancer cells. Valproate inhibited the induction of severalantiviral genes after oncolytic HSV infection, and resulted in enhancedviral propagation in glioma cells, even in the presence of IFN (Otsuki,et at, Mol. Ther., 16:1546-1555 (2008)). Importantly, valproatetreatment had no augmenting effect on viral yield in normal humanastrocytes. Valproate pretreatment was also shown to enhance HSVpropagation in tumors 10-fold in vivo and improved the survival of nudemice bearing U87delta-EGFR brain tumors.

Therefore, in some embodiments, the virus is administered in combinationwith an HDAC inhibitor. In some embodiments, the virus is administeredin combination with valproate, the vacccinia protein B18R, Jak inhibitor1, or vorinostat.

Other immunosuppressants such as cyclosporin, prednisone, dexamethasone,or other steroidal anti-inflammatory, can also be used to reduce theimmune response immediately before, during, or shortly afteradministration of the therapeutic virus. The immunosuppressant is thendiscontinued or decreased to allow the patient's immune system toprevent inflammation and/or killing of the virus after it has competedthe desired killing of tumor or diseased tissue.

Suitable immunosuppressants are known in the art and includeglucocorticoids, cytostatics (such as alkylating agents,antimetabolites, and cytotoxic antibodies), antibodies (such as thosedirected against T-cell recepotors or I1-2 receptors), drugs acting onimmunophilins (such as cyclosporine, tacrolimus, and sirolimus) andother drugs (such as interferons, opioids, TNF binding proteins,mycophenolate, and other small molecules such as fingolimod). The dosageranges for immunosuppressant agents are known in the art. The specificdosage will depend upon the desired therapeutic effect, the route ofadministration, and on the duration of the treatment desired. Forexample, when used as an immunosuppressant, a cytostatic maybeadministered at a lower dosage than when used in chemotherapy.Immunosuppressants include, but are not limited to, FK506, prednisone,methylprednisolone, cyclophosphamide, thalidomide, azathioprine, anddaclizumab, physalin B, physalin F, physalin G, seco-steroids purifiedfrom Physalis angulata L., 15-deoxyspergualin, MMF, rapamycin and itsderivatives, CCI-779, FR 900520, FR 900523, NK86-1086, depsidomycin,kanglemycin-C, spergualin, prodigiosin25-c, cammunomicin, demethomycin,tetranactin, tranilast, stevastelins, myriocin, gliotoxin, FR 651814,SDZ214-104, bredinin, WS9482, mycophenolic acid, mimoribine,misoprostol, OKT3, anti-IL-2 receptor antibodies, azasporine,leflunomide, mizoribine, azaspirane, paclitaxel, altretamine, busulfan,chlorambucil, ifosfamide, mechlorethamine, melphalan, thiotepa,cladribine, fluorouracil, floxuridine, gemcitabine, thioguanine,pentostatin, methotrexate, 6-mercaptopurine, cytarabine, carmustine,lomustine, streptozotocin, carboplatin, cisplatin, oxaliplatin,iproplatin, tetraplatin, lobaplatin, JM216, JM335, fludarabine,aminoglutethimide, flutamide, goserelin, leuprolide, megestrol acetate,cyproterone acetate, tamoxifen, anastrozole, bicalutamide,dexamethasone, diethylstilbestrol, bleomycin, dactinomycin,daunorubicin, doxirubicin, idarubicin, mitoxantrone, losoxantrone,mitomycin-c, plicamycin, paclitaxel, docetaxel, topotecan, irinotecan,9-amino camptothecan, 9-nitro camptothecan, GS-211, etoposide,teniposide, vinblastine, vincristine, vinorelbine, procarbazine,asparaginase, pegaspargase, octreotide, estramustine, and hydroxyurea,and combinations thereof. Preferred immunosuppressants willpreferentially reduce or inhibit the subject's immune response, withoutreducing or inhibiting the activity of the virus.

IV. Kits

Dosage units including virus in a pharmaceutically acceptable carrierfor shipping and storage and/or administration are also disclosed.Active virus should be shipped and stored using a method consistent withviability such as in cooler containing dry ice so that viruses aremaintained below 4° C., and preferably below −20° C. VSV virus shouldnot be lyophilized. Components of the kit may be packaged individuallyand can be sterile. In one embodiment, a pharmaceutically acceptablecarrier containing an effective amount of virus is shipped and stored ina sterile vial. The sterile vial may contain enough virus for one ormore doses. Virus may be shipped and stored in a volume suitable foradministration, or may be provided in a concentrated titer that isdiluted prior to administration. In another embodiment, apharmaceutically acceptable carrier containing an effective amount ofvirus can be shipped and stored in a syringe.

Typical concentrations of concentrated viral particles in the sterilesaline, phosphate buffered saline or other suitable media for the virusis in the range of 10⁸ to 10⁹ with a maximum of 10¹². Dosage unitsshould not contain membrane disruptive agents nor should the viralsolution be frozen and dried (i.e., lyophilized), which could kill thevirus.

Kits containing syringes of various capacities or vessels withdeformable sides (e.g., plastic vessels or plastic-sided vessels) thatcan be squeezed to force a liquid composition out of an orifice areprovided. The size and design of the syringe will depend on the route ofadministration. For example, in one embodiment, a syringe foradministering virus intratumorally, is capable of accurately deliveringa smaller volume (such as 1 to 100 μl). Typically, a larger syringe,pump or catheter will be used to administer virus systemically. Any ofthe kits can include instructions for use.

V. Methods of Manufacture

A. Engineering Recombinant VSV Viruses

The native VSV genome is a single negative-sense, non-segmented stand ofRNA that contains five genes (N, L, P, M, and G) and has a total size of11.161 kb. Methods of engineering recombinant viruses by reconstitutingVSV from DNA encoding a positive-sense stand of RNA are known in the art(Lawson, et al., PNAS, 92:4477-4481 (1995), Dalton and Rose, Virology.,279:414421 (2001)). For example, recombinant DNA can be transcribed byT7 RNA polymerase to generate a full-length positive-strand RNAcomplimentary to the viral genome. Expression of this RNA in cells alsoexpressing the VSV nucleocapsid protein and the two VSV polymerasesubunits results in production of VSV virus (Lawson, et al., PNAS,92:4477-4481 (1995)). In this way, VSV viruses can be engineered toexpress variant proteins, additional proteins, foreign antigens,targeting proteins, or therapeutic proteins using known cloning methods.Methods of preparing exemplary suitable VSV viruses where the geneencoding the VSV G protein is deleted and replaced with a gene encodingthe Lassa virus glycoprotein are described in more detail above.

In some embodiments, the chimeric VSV is prepared by substituting thesequence encoding the G protein on the plasmid referred as VSVXN2(Schnell, et al., J. Virol., 70:2318-2323 (1996)) with a heterologousglycoprotein, such as the glycoprotein from Lassa virus.

In other embodiments the chimeric VSV is prepared by substituting thesequence encoding the G protein on plasmid pVSV(+) described in Whelan,et al., Proc. Natl. Acad. Sci. U.S.A., 92(18):8388-92 (1995). Whelandescribes the constructions of a full-length cDNA clone of VSV assembledfrom clones of each of the VSV genes and intergenic junctions. Theseclones were assembled into a full-length cDNA and inserted in bothorientations between the bacteriophage T7 promoter and a cDNA copy ofthe self-cleaving ribozyme from the antigenomic strand of HDV. Theresulting plasmids were named pVSV1(+) and pVSV1(−) to reflect thepolarity of the T7 transcript they generated: VSV antigenomic or genomicRNA, respectively.

The T7 transcripts contained two non-VSV nucleotides (GG) at their 5′ends but were cleaved by the HDV ribozyme to generate a 3′ terminuswhich corresponded precisely to the 3′ end of the VSV antigenomic orgenomic sequence, an important requirement for VSV RNA replication.Transfection of plasmids into BHK21 cells infected with vTF7-3 wasperformed under the conditions and with quantities of support plasmidsas described (Pattnaik, et al., Cell, 69:1011-1020 (1992)), and up to 5ug of pVSV1(+) or pVSV1(−). Transfected cells were incubated at 31° C.or 37° C. For some experiments, pVSV1(+) and pVSV1(−) were linearized bydigestion at a unique Nhe I site located downstream of the T7 terminatorin the pGEM-3-based plasmids.

To identify cDNA-derived virus unambiguously, several genetic markerswere incorporated into the full-length cDNA clones. All five genes wereof the Indiana serotype of VSV, but whereas the N, P, M, and L genesoriginated from the San Juan strain, the G gene was from the Orsaystrain. In addition, the functional P clone has 28 nucleotide sequencedifferences from the published San Juan sequence and in the case ofpVSV1(+) the 516 nt at the 5′ end of the VSV genome originated from pDI,the clone of DI-T RNA (Pattnaik, et al., Cell, 69:1011-1020 (1992)).

B. Creating Mutant VSV Virus

RNA viruses are prone to spontaneous genetic variation. The mutationrate of VSV is about 10⁴ per nucleotide replicated, which isapproximately one nucleotide change per genome (Drake, et al., Proc.Natl. Acad. Sci. USA, 96:13910-13913). Therefore, mutant VSV virusesexhibiting desired properties can be developed by applying selectivepressure. Methods for adaptation of VSV viruses through repeatedpassaging is described in the art. See, for example, Wollmann, et al.,J. Virol., 79(10): 6005-6022 (2005). Selective pressure can be appliedby repeated passaging and enhanced selection to create mutant virus withdesirable traits such as increased infectivity and oncolytic potentialfor a cell type of interest. The cell type of interest could be general,such as cancer cells, or specific such as glioblastoma cells. Mutantvirus can also be selected based on reduced toxicity to normal cells.Methods of enhanced selection include, but are not limited to, shorttime for viral attachment to cells, collection of early viral progeny,and preabsorption of viral particles with high affinity of undesirablecells (such as normal cells). Mutations can be identified by sequencingthe viral genome and comparing the sequence to the sequence of theparental strain.

DNA encoding the VSV genome can also be used as a substrate for randomor site directed mutagenesis to develop VSV mutant viruses. Mutagenesiscan be accomplished by a variety of standard, mutagenic procedures.Changes in single genes may be the consequence of point mutations thatinvolve the removal, addition or substitution of a single nucleotidebase within a DNA sequence, or they may be the consequence of changesinvolving the insertion or deletion of large numbers of nucleotides.

Mutations can arise spontaneously as a result of events such as errorsin the fidelity of nucleic acid replication or the movement oftransposable genetic elements (transposons) within the genome. They alsoare induced following exposure to chemical or physical mutagens. Suchmutation-inducing agents include ionizing radiations, ultraviolet lightand a diverse array of chemicals such as alkylating agents andpolycyclic aromatic hydrocarbons all of which are capable of interactingeither directly or indirectly (generally following some metabolicbiotransformations) with nucleic acids. The nucleic acid lesions inducedby such environmental agents may lead to modifications of base sequencewhen the affected DNA is replicated or repaired and thus to a mutation.Mutation also can be site-directed through the use of particulartargeting methods. Various types of mutagenesis such as randommutagenesis, e.g., insertional mutagenesis, chemical mutagenesis,radiation mutagenesis, in vitro scanning mutagenesis, random mutagenesisby fragmentation and reassembly, and site specific mutagenesis, e.g.,directed evolution, are described in U.S. Patent Application No.2007/0026012.

Mutant viruses can be prepared by site specific mutagenesis ofnucleotides in the DNA encoding the protein, thereby producing DNAencoding the mutant. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis and PCR mutagenesis. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once. Insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Substitutions, deletions, insertionsor any combination thereof can be combined to arrive at a fmalconstruct. The mutations must not place the sequence out of readingframe and preferably will not create complementary regions that couldproduce secondary mRNA structure. Substitution variants are those inwhich at least one residue has been removed and a different residueinserted in its place.

EXAMPLES Example 1 Chimeric Lassa-VSV Virus Infected and Killed Gliomaswithout Causing Neurological Dysfunction Materials and Methods

Viruses and Cells

VSV-wtG and chimeric VSV's expressing the G protein from either Lassafever virus (VSV-LASV-G), rabies virus (VSV-RABV), lymphocyticchoriomeningitis virus (VSV-LCMV-G), Ebola virus (VSV-EBOV-G), orMarburg virus (VSV-MARV-G) were generated as described previously(Beier, et al., Proc. Natl. Acad. Sci. USA, 108:15414-15419 (2011); Jae,et al., Science, 344:1506-1510 (2014); Krishnan, et al., Viruses,4:2471-2484. A GFP reporter gene was engineered into the first genomeposition of these VSVs. VSV-EBOV-G and VSV-LASV-G that did not expressany reporter genes were also tested in some assays (Garbutt, et al., JVirol., 78(10):5458-65 (2004)).

VSV-EBOV

The EBOV glycoprotein sequence expressed in VSV-EBOV is derived from theMayinga strain of Zaire Ebola virus as described in the methods ofGarbutt, et al. (2004) J. Virol 78: 5458-5465. The nucleotide sequenceshown below was determined by direct sequencing of VSV-EBOV samples andmatches the sequence for the full-length (non-secreted) glycoproteingene found in GenBank accession NC_002549 nt 6039-8068. Note that theGenBank sequence over this region is 2030 nt long, whereas the sequencebelow is 2031 nt in length. This difference derives from the fact thatthe GenBank sequence is based on the genomic RNA sequence, whereas thesequence below is based on the mRNA sequence that has been ‘edited’ bythe viral polymerase to include an extra ‘A’ nucleotide between6918-6924 of the GenBank sequence.

VSV-EBOV Glycoprotein Gene Nucleotide Sequence (2031 nt)

(SEQ ID NO: 6) ATGGGCGTTACAGGAATATTGCAGTTACCTCGTGATCGATTCAAGAGGACATCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCACTTGGAGTCATCCACAATAGCACATTACAGGTTAGTGATGTCGACAAACTAGTTTGTCGTGACAAACTGTCATCCACAAATCAATTGAGATCAGTTGGACTGAATCTCGAAGGGAATGGAGTGGCAACTGACGTCCCATCTGCAACTAAAAGATGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGAAGCTGGTGAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCTGACGGGAGTGAGTGTCTACCAGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGGTATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGACTTTGCCTTCCATAAAGAGGGTGCTTTCTTCCTGTATGATCGACTTGCTTCCACAGTTATCTACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGCCCCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTCAATGCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAGGCTACCGGTTTTGGAACCAATGAGACAGAGTACTTGTTCGAGGTTGACAATTTGACCTACGTCCAACTTGAATCAAGATTCACAGCACAGTTTCTGCTCCAGCTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCACGGGAATACTAATTTGGAAGGTCAACCCCGAAATTGATACAACAATCGGGGAGTGGGCCTTCTGGGAAACTAAAAAAAACCTCACTAGAAAAATTCGCAGTGAAGAGTTGTCTTTCACAGTTGTATCAAACGGAGCCAAAAACATCAGTGGTCAGAGTCCGGCGCGAACTTCTTCCGACCCAGGGACCAACACAACAACTGAAGACCACAAAATCATGGCTTCAGAAAATTGCTCTGCAATGGTTCAAGTGCACAGTCAAGGAAGGGAAGCTGCAGTGTCGCATCTAACAACCCTTGCCACAATCTCCACGAGTCCCCAATCCCTCACAACCAAACCAGGTCCGGACAACAGCACCCATAATACACCCGTGTATAAACTTGACATCTCTGAGGCAACTCAAGTTGAACAACATCACCGCAGAACAGACAACGACAGCACAGCCTCCGACACTCCCTCTGCCACGACCGCAGCCGGACCCCCAAAAGCAGAGAACACCAACACGAGCAAGAGCACTGACTTCCTGGACCCCGCCACCACAACAAGTCCCCAAAACCACAGCGAGACCGCTGGCAACAACAACACTCATCACCAAGATACCGGAGAAGAGAGTGCCAGCAGCGGGAAGCTAGGCTTAATTACCAATACTATTGCTGGAGTCGCAGGACTGATCACAGGCGGGAGAAGAACTCGAAGAGAAGCAATTGTCAATGCTCAACCCAAATGCAACCCTAATTTACATTACTGGACTACTCAGGATGAAGGTGCTGCAATCGGACTGCCCTGGATACCATATTTCGGGCCAGCAGCCGAGGGAATTTACATAGAGGGGCTAATGCACAATCAAGATGGTTTAATCTGTGGGTTGAGACAGCTGGCCAACGAGACGACTCAAGCTCTTCAACTGTTCCTGAGAGCCACAACTGAGCTACGCACCTTTTCAATCCTCAACCGTAAGGCAATTGATTTCTTGCTGCAGCGATGGGGCGGCACATGCCACATTCTGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCAGGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGGCGTTATAATTGCAGTTATCGCTTTATTCTGTATATGC AAATTTGTCTTTTAG

VSV-EBOV Glycoprotein Amino Acid Sequence (676 a.a.)

(SEQ ID NO: 7) MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCIC KFVF

VSV-1′GFP-EBOV

The EBOV glycoprotein sequence expressed in VSV-1′GFP-EBOV is derivedfrom the Mayinga strain of Zaire Ebola virus as described in the methodsof Wong, et al. (2010) J. Virol 84: 163-175. The nucleotide sequenceshown below was determined by direct sequencing of VSV-1′GFP-EBOVsamples and is similar (but not identical) to the sequence for thefull-length (non-secreted) glycoprotein gene found in GenBank accessionAF086833 nt 6039-8068. The mucin domain (a.a. 309-489) has been deletedfrom the protein and two amino acids are different (N40S and I662V)relative to the GenBank sequence. Whereas the mucin deletion and 1662Vmutations were intentionally introduced and are described in the methodsof Wong, et al. (2010), the N40S mutation was not described and it isunknown whether this mutation existed within the original recombinantplasmid used to generate the virus or emerged after recovery throughrepeated passages.

VSV-1′GFP-EBOV Glycoprotein Gene Nucleotide Sequence (1494 nt)

(SEQ ID NO:8)ATGGGCGTTACAGGAATATTGCAGTTACCTCGTGATCCATTCAAGAGGACATCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCACTTGGAGTCATCCACAGTAGCACATTACAGGTTAGTGATGTCGACAAACTAGTTTGTCGTGACAAACTGTCATCCACAAATCAATTGAGATCAGTTGGACTGAATCTCGAAGGGAATGGAGTGGCAACTGACGTGCCATCTGCAACTAAAAGATGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGAAGCTGGTGAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCTGACGGGAGTGAGTGTCTACCAGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGGTATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGACTTTGCCTTCCATAAAGAGGGTGCTTTCTTCCTGTATGATCGACTTGCTTCCACAGTTATCTACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGCCCCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTCAATGCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAGGCTACCGGTTTTGGAACCAATGAGACAGAGTACTTGTTCGAGGTTGACAATTTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCTGCTCCAGCTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCACGGGAAAACTAATTTGGAAGGTCAACCCCGAAATTGATACAACAATCGGGGAGTGGGCCTTCTGGGAAACTAAAAAAAACCTCACTAGAAAAATTCGCAGTGAAGAGTTGTCTTTCTCTAGAGCAGGACTGATCACAGGCGGGAGAAGAACTCGAAGAGAAGCAATTGTCAATGCTCAACCCAAATGCAACCCTAATTTACATTACTGGACTACTCAGGATGAAGCTGCTGCAATCGGACTGGCCTGGATACCATATTTCGGGCCAGCAGCCGAGGGAATTTACATAGAGGGGCTAATGCACAATCAAGATGGTTTAATCTGTGGGTTGAGACACCTGGCCAACGAGACGACTCAAGCTCTTCAACTGTTCCTGAGAGCCACAACTGAGCTACGCACCTTTTCAATCCTCAACCGTAAGGCAATTGATTTCTTGCTGCAGCGATGGGGCGCCACATGCCACATTCTGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCAGGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGGCGTTGTAATTGCAGTTATCGCTTTATTCTGTATATCCAAATTTGTCTTTTAG

VSV-1′GFP-EBOV Glycoprotein Amino Acid Sequence (497 a.a.)

(SEQ ID NO: 9)MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHSSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIRYGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFSRAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVVIAVIALFCICKFVF

VSV-LASV

The viral genome is a VSV background where the sequence encoding the VSVG protein is substituted for a sequence encoding the glycoprotein fromLassa virus, and a sequence encoding GFP is inserted into the firstposition of the viral genome. The LASV glycoprotein sequence expressedin VSV-LASV is derived from the Josiah strain of Lassa virus asdescribed in the methods of Garbutt, et al. (2004) J. Virol 78:5458-5465.

VSV-1′GFP-LASV

The LASV glycoprotein sequence expressed in VSV-1′GFP-LASV is derivedfrom the Josiah strain of Lassa virus as described in the methods ofJae, et al. (2013) Science 340: 479-483.

Viral infection was detected by the GFP reporter, using fluorescent andphase contrast imaging under a microscope. All viruses were used at anMOI of 0.1; thus Lassa-VSV replicated in gliomas in order to infect allcells tested.

For in vitro experiments, viruses were propagated on Vero cells,purified, and concentrated using sucrose gradient centrifugation(Cureton, et al., PLoS Pathog, 6, e1001127 (2010). For in vivoexperiments, viruses were propagated on Vero cells and filter-purifiedaccording to a protocol described previously (Lawson, et al., Proc.Natl. Acad. Sci. USA, 92:4477-4481 (1995). All viruses wereplaque-titered on Vero cells prior to experiments. Human glioma U87 andU118 were obtained from ATCC (Manassas, Va.), mouse glioma CT2A was agift from Dr T Seyfried (Boston College, Chestnut Hill, Mass.), humanmelanoma YUMAC and normal human melanocytes were provided by Yale SPOREin Skin Cancer. Normal human glial cells were derived from humantemporal lobectomies (Ozduman, et al., J. Neurosci, 28:1882-1893(2008)). Normal human dermal fibroblasts were purchased from Cambrex(Walkersville, Md.). Normal human embryonic neurons were purchased fromSciencell (Carlsbad, Calif.). The human cancer cell lines SJSA-1,BT-549, T-47D, HCT116, SW480, T24, and RT4, and DU-145 lines were kindlyprovided. Stably transfected tumor cells expressing red fluorescentprotein RFP (rU87 and rYUMAC) were generated as described earlier(Wollmann, et al., J. Virol., 87:6644-6659 (2013)). Primary cultures ofmouse brain were generated by dissociating cortex of E17 mice forpredominantly neuronal cultures and whole brain tissue of P1 mice formixed neuronal/glia cultures. Cells were plated in MEM (Invitrogen,Carlsbad, Calif.) supplemented with 10% FBS overnight before medium wasreplaced with Neurobasal/B27 medium (Invitrogen). Melanoma cells weremaintained in Opti-MEM/5% FBS, melanocyte medium containing additionalsupplements listed elsewhere (Wollmann, et al., J. Virol., 87:6644-6659(2013)). Glioma cells, human glia and fibroblasts were maintained inMEM/10% FBS (Invitrogen). All cultures were kept in humidifiedatmosphere containing 5% CO₂ at 37° C.

In Vitro Experiments

Viral infection of mixed neuronal/glial cultures, and U87, U118, CT2A,and YUMAC tumor cultures was monitored by quantifying GFP expression ofinfected cells compared to total number of cells on at least 10microscopic fields using fluorescence microscopy. Cultures were assessedfor presence of cytopathic effects before and after virus application.Cytopathic effects were noted as the appearance of rounding, blebbingand syncitia formation. For analysis of infection characteristic ofchimeric VSVs on mixed neuronal cultures, morphology of infected cellswas used as a guide for identifying neuronal or glia infection by thevirus. Identification of cell type was later corroborated byimmunohistochemistry for NeuN and GFAP. Mixed human brain cultures wereestablished by first plating a glia monolayer. Two days later, humanneurons were seeded onto the glia monolayer. After 7 days in culture andmorphological confirmation of neuron process outgrowth, cultures wereinoculated with VSV-wtG or VSVLASV-G (MOI 1). GFP expression wasquantified 24 hours later.

Plaque assay was used to assess viral replication. In short, cells wereinfected at an MOI of 0.1, residual virus was removed by replacingsupernatant, and progeny virus was collected from the supernatant at theindicated time points. Serial dilutions were plated on monolayers ofVero or BHK cells, respectively. 2.5% Agar solution in MEM was used assemi-solid overlay. For IFN pretreatment, cultures were incubated for 8hours with recombinant hybrid interferon type 1 IFNαA/D (Sigma-Aldrich;catalog no. I4401) at a concentration of 100 IU/ml. To testgeneralization of the oncolytic effect of VSV-LASV-G to other humancancer types, 8 human tumor cell types were infected at an MOT 3(primary inoculation). 2 hours later inoculum was removed and cultureswere washed 3 times with PBS before addition of fresh medium. 24 hourslater infection rates were determined by GFP expression. To assess thecapability of VSV-LASV-G to propagate in these tumor cultures the 24 hpisupernatant was filtered to remove cellular debris and transferred ontoan uninfected monolayer of the same tumor designation (secondaryinoculation).

Image Analysis

Virus infected cultures and histological sections of mouse brain wereanalyzed using a fluorescence microscope (IX 71, Olympus Optical, Tokyo,Japan). A fluorescence stereomicroscope (SZX12, Olympus Optical) wasused for whole brain scanning before sectioning.

Results

To test the efficacy of different binding glycoproteins, 6 recombinantVSVs were compared, each expressing a different binding glycoprotein(FIGS. 1A-1C). Five chimeric VSVs were tested in which the VSVglycoprotein was replaced (Beier, et al., Proc. Natl. Acad. Sci. USA,108:15414-15419 (2011); Jae, et al., Science, 340:479-483 (2013);Krishnan, et al., Viruses, 4:2471-2484 (2012)) by the G genes from otherviruses, including Lassa fever, rabies, lymphocytic choriomeningitis,Ebola, or Marburg virus. These chimeric viruses were compared withcontrol VSVs that retained the normal VSV glycoprotein. Chimericviruses, which like VSV-wtG, also encoded GFP in the first genomicposition, were tested first in human and mouse glioblastoma (GBM). Allsix viruses infected GBM cells in vitro FIG. 1A), with a greater levelof infection of human GBM (U87 and U118) than mouse GBM (CT2A) 24 hpi(FIG. 1A). Generally speaking, Lassa-VSV showed the most robustinfection of the 3 gliomas tested (U87, U118, CT2A) at 24 hrs, andearlier, while other viruses also infected some of the gliomas, but notall of them. Specifically, in U87 glioma, all viruses showed goodinfection except VSV-MARV (FIG. 1A). In U118 glioma, VSV-LASV, VSV-EBOV,and VSV-RABV showed high levels of infection, whereas VSV-MARV andVSV-LCMV significantly less. In mouse CT-2A glioma, VSV-LASV performedthe best; VSV-EBOV infected almost as well as VSV-LASV (FIG. 1A).Lassa-VSV (VSV-La-G) infected and killed 100% of two human gliomas, U87and U118.

All six viruses replicated on human GBMs, as seen at 24 hrs postinoculation (hpi) (FIG. 1B). Typical of VSV-infected cells (Ozduman, etal., J. Neurosci, 28:1882-1893 (2008); Wollmann, et al., J. Virol,81:1479-1491 (2007)), infected cells died, as confirmed by staining withmembrane impermeant dyes.

The relative infection of all six viruses on mouse and human braincultures that included neurons and glia was also tested. The relativeinfection of mouse neurons vs glia is shown in FIG. 1D. VSV-wtG showedthe greatest level of neuronal infection (around 90%). VSV-LASV-G andVSV-LCMV-G showed the least neuronal infection. When plaque size wasexamined as a measure of infectivity and replication, VSV-LASV plaqueswere bigger than VSV-LCMV and VSV-EBOV on human gliomas U87 and U118(FIG. 1C), indicating enhanced infection and replication. VSV generatedthe largest plaques on tumor cells, but also showed large plaques onnormal brain cells, an unwanted phenotype. Among the chimeric viruses,VSV-LASV and VSV-EBOV generated smaller plaques on human brain thanVSV-wtG and VSV-LCMV (FIG. 1C), and showed much weaker infection of purehuman neuronal cultures than VSV-wtG (FIG. 3A).

Lassa-VSV, which was the superior oncolytic candidate from an analysisof glycoproteins from 5 unrelated viruses that were incorporated intothe VSV genome, was also compared with other VSVs that expressed thenormal VSV glycoprotein. Of 39 potential oncolytic viruses, including 17variants of VSV, (Wollmann, et al., J Virol., 79: 6005-6022 (2005);Wollmann, et al., J Virol., 81:1479-1491 (2007); Wollmann, et al., JVirol., 84:1563-73 (2010); Wollmann, et al., Cancer J., 18:69-81 (2012);Paglino, et al., J. Virol., 85:9346-58 (2011); Ozduman, et al., J.Virology, 83:11540-11549 (2009); Ozduman, et al., J Neurosci.,28:1882-1893 (2008)), Lassa-VSV was identified as a superior candidatein terms of a strong safety profile, as well as an effective potentialto selectively target and kill GBMs and to precipitate an immune attackon the glioma.

Together, the in vitro data point to VSV-LASV as having the bestcombination of glioma infectivity (FIGS. 1A and 1C) and low neuronaltropism (FIG. 1D). VSV-EBOV was selected as a second candidate toinclude for in vivo testing being another virus with broad gliomatropism and evidence of reduced neurotropsim compared to VSVwtG in bothmouse and human brain, although not as reduced as VSV-LASV. VSV-Lassashowed the least infection of neurons, coupled with a strong infectionof human glioma. In contrast, a control rabies-VSV chimeric virus showedstrong infection of neurons and would be a relatively very poorcandidate for glioma treatment. Recombinant VSVs with minor changes tothe genome, for instance VSV-M51, can potentially revert to the moreaggressive wild type virus by gene mutation. In contrast, since theentire binding of VSV to cells has been switched to Lassa glycoprotein,and the VSV glycoprotein gene is deleted, this virus cannot revert tothe wild type VSV, a substantial benefit. A primary mechanism oftargeting is the absence or attenuation of the intrinsic anti-viralresponse in the majority of cancer cells, including gliomas, compared tonormal cells that have an intrinsic interferon mediated antiviralresponse to VSV and other viruses (Stojdl, et al., Nat. Med., 6:821-825(2000); Stojdl, et al., Cancer Cell., 4:263-275 (2003); Wollmann, etal., J. Virol., 81:1479-1491 (2007)).

Furthermore, although there is not full agreement as to the origin ofcancer stem cells, with some indications that the cells initiate cancer,or that they arise from multiple mutations in dividing tumor cells,there is more agreement that cells that express antigens typical of stemcells do show resistance to small-molecule cancer therapeutics, and toradiation treatment. Glioma stem cells are reported to express CD133 andnestin. In preliminary experiments, using cells expressing CD133,Lassa-VSV was found to infect and kill these stem-like cells in culture,indicating another potential attribute of the use of this virus in thetreatment of cancer.

Example 2 Lassa-VSV is Safe in the Brain Materials and Methods

Mouse Procedures

6-8 week old male Swiss Webster mice received the following virus doses:I.C.: 3.6×10⁴ pfu in 1 ml into the right striatum (2 mm lateral, 0.5 mmrostral to Bregma at 3 mm depth), I.V.: 10⁶ pfu in 100 μl via tail veininjection, I.N.: 2.5×10⁵ pfu in 25 μl in each nostril. Stereotacticapplication of virus or tumor cells was performed under full anesthesiausing ketamine/xylazine (100 and 10 mg/kg, respectively) applied viai.p. route. Uni- and bilateral intracranial glioma and melanomaxenografts were established in 4-6 week old male CB17 SCID mice byinjection of 5×10⁴ cells each into the left and right striatum (Ozduman,et al., J Neurosci, 28:1882-1893 (2008); Wollmann, et al., J Virol,87:6644-6659 (2013)). 15 days post tumor placement mice received viruseither via a unilateral intratumoral injection (3.6×10⁴ pfu in 1 μl) orvia tail vein injection (10⁶ pfu in 100 μl). Mice were monitored dailyand sacrificed if one of the following conditions were observed: A)weight loss of 25% or more, B) immobility, C) occurrence of adverseneurological symptoms, or D) reaching the end of the observation periodof the survival study.

For histologic analysis of early states of viral infection mice weresacrificed at 2 or 8 days post viral inoculation. After overdose withanesthetic, mice were transcardially perfused with 4% paraformaldehyde.Brains were harvested and stored in 4% paraformaldehyde, dehydrated in30% sucrose solution and cut in 20 to 30 μm coronal sections with acryostat. For detection of live virus in designated mice after short (2days) and long (>60 days) exposure to VSV-LASV-G, tissue samples werecollected under sterile conditions from brain, lung, blood, and liverafter euthanasia. Tissues were mechanically homogenized in PBS using amicrocentrifuge tube tissue grinder. Part of the resultant tissuesuspension was plated onto BHK monolayers and assessed for presence ofGFP positive cells 24 hours later. To test the capability of VSV-LASV-Gto induce antibody production, adult Swiss Webster mice received anintranasal and intramuscular primary VSV-LASV-G inoculation (atconcentrations listed above) followed by a boost 4 weeks later. 2 weekslater, mice were euthanized, bled, and serum was collected. Antibodycontaining serum was diluted 1:50 to 1:10,000.

Brain sections from transgenic mice expressing GFP in hypocretin neuronswere used to target GFP. Brain sections of IFNα/β-R knock-out miceinfected with VSV-LASV-G were used to target VSV-LASV-G expression. rU87and rYUMAC cells were tested for pathogens before tumor grafting andfound to be pathogen free. All animal experiments adhered toinstitutional guidelines and were approved by the Yale University AnimalCare and Use Committee.

Rat Procedures

Safety of intracranial VSV-LASV-G in rats was tested. Stereotacticcoordinates and applied virus dose equaled those in rat tumor modelsdescribed in the following. Syngeneic brain tumor models wereestablished via stereotactic injection of 50,000 cells in 3 μlsuspension into the right striatum (0.7 mm rostral of Bregma, 3.5 mmlateral, 5 mm deep) of 7-8 week old Lewis rats (rat CNS-1 glioma). 7days post tumor placement, rats received a single intracranial injectionof 3 μl suspension containing 1.2×105 PFU of VSV-LASV-G into the area ofthe tumor. Rats were euthanized at 3 dpi and brains harvested.

Intrcranial Injection Safety Assays

The Lassa-VSV and Ebola-VSV viruses were injected directly into thebrains of 6 week old Swiss-Webster mice (n=10 mice for each virus) usingstereotactic injection procedures. A Hamilton syringe controlled by aStoelting stereotactic injector (Stoelting, Wood Dale, Ill.) was used toinject 1 d of virus solution into the striatum (2 mm lateral, 0.4 mmrostral to bregma, at 3 mm depth). As a control, attenuated VSV-1′GFPand attenuated VSV-M51 viruses were injected directly into the brains ofmice (n=6 mice for each virus) using the same procedure. All injectionswere 3.6×10⁴ pfu in 1 d. Animals were monitored daily for body weight,grooming, and overall health. Mice were euthanized with a pentobarbitaloverdose when neurological symptoms were detected or when body weightdropped below 75%.

Statistical Analysis

Statistical significance was determined by Student's t-test, ANOVA, andChi-Square test. P-values <0.05 were considered statisticallysignificant. Survival studies were non-blinded. Mice were allocated toexperimental or control group based on cage number. Power analysis wasemployed to determine group size for survival experiments oftumor-bearing mice and of virus injected mice.

Results

To corroborate further the relative lack of infection of neurons of thechimeric viruses, VSV-LASV and VSV-EBOV were compared to two VSVs thatexpressed the VSV glycoprotein in vivo; one of the VSVs was furtherattenuated by including an M51 mutation which enhances the antiviralinnate immune response against the virus (Waibler et al, 2007).Injection of either VSV with normal wild-type G (VSV-wtG) into thenormal brain generated lethal consequences, with a median survival of3.5 days for VSV-wtG (n=6) and 8 days for the attenuated strain VSV-M51(n=6) (FIG. 2A) consistent with previous observations (Ozduman et al.,2009). This underlines the neurotoxicity of VSV with normal VSV-Gprotein, even when attenuated. In striking contrast, direct injectionsof VSV-LASV-G or VSV-EBOV-G, (3.6×10⁴ pfu in 1 μl) into normal mousebrains exerted no adverse effect in >112 days (n=8 each virus; p<0.001;Chi square test) (FIG. 2A).

In another experiment VSV-LASV-G was compared with VSV-IFN, anIFN-expressing virus modeled after one (Obuchi, et al., J. Virol.,77:8843-8856 (2003) currently in clinical trials for the treatment ofliver cancer (NCT01628640). All mice (n=9) receiving intracranialVSV-IFN died within 12 days consistent with reports of this virusinfecting brain meninges (Yarde, et al., Cancer Gene Ther., 20:616-621(2013); all mice that received VSV-LASV-G (n=4 here) survived with noadverse side effects (FIG. 2B). Similarly, VSV-LASV-G injected into therat brain also showed no sign of neurotoxicity (>80 days, n=3). No virusor infected cells were detected in the brain or elsewhere (liver,spleen, blood) by histological analysis or culture of inoculated mousetissue at the conclusion of the experiment, indicating the totalelimination of the virus. The injections of VSV-LASV-G and VSV-EBOV-Gwere viable, as euthanasia at 2 dpi revealed limited infection of gliawithin the brain. However, by 8 dpi, few or no infected cells could befound. VSV-LASV-G and VSV-EBOV-G were also injected intravenously (n=5each), and an additional set of mice were inoculated at combinedintranasal/intramuscular/subcutaneous (n=5, total 8×10⁶ pfu VSV-LASV-G)sites; none of these mice showed signs of viral pathogenicity and novirus could be harvested from these mice 2 months postinoculation.

A different VSV-LASV-G that contained no GFP (Garbutt, et al., J.Virol., 78:5458-5465 (2004) was also tested and found to have no adverseeffect after injection into the mouse brain (n=5) and followed for 6weeks, showing that the attenuation mediated by inclusion of the GFPgene in the first genomic position was not critical for CNS safety. The100% survival of mice injected with VSV-LASV-G and VSV-EBOV-G correlatedwith a very low infectivity of these viruses in pure neuronal cultures,particularly when compared to VSV with the normal VSV G protein (FIG.2A-2B).

The most problematic aspect of using VSV either as an oncolytic virus oras a vaccine vector against more dangerous viruses including Ebola(Garbutt, et al., J. Virol., 78:5458-5465 (2004), HIV, and otherpathogenic viruses, has been the concern about adverse effects of thevirus in the brain (Ozduman, et al., J. Virol., 83:11540-11549 (2009);Huneycutt, et al., Brain Res., 635:81-95 (1994)). A number of previousstudies have described the potential for VSV to selectively target anddestroy many different types of tumors. However, a substantial problemwith the use of VSV as an oncolytic virus, both outside and within thebrain, has been the possibility of adverse effects within the brainpotentially leading to motor dysfunction (Huneycutt, et al., Brain Res.,635:81-95 (1994), behavioral disturbances (Lundh, et al., J.Neuropathol. Exp. Neurol., 47:497-506 (1988) or death (Huneycutt, etal., Brain Res., 635:81-95 (1994); van den Pol, et al., J. Virol,76:1309-1327 (2002). The data show that eliminating the nativeglycoprotein from the VSV genome and substituting binding glycoproteinsfrom other viruses greatly reduces infection and cytolysis of neurons.All five chimeric VSVs tested showed considerably reduced neuron tropismand replication compared with the natural VSV glycoprotein. Not onlywere the chimeric viruses tested safe within the brain, but directinjection of VSV-LASV-G into the brains of SCID mice lacking the normalcomplement of B and T immune cells generated no adverse effect, and theinnate immune system within the brain eliminated the virus.

Importantly, after intravenous inoculation, the chimeric virusesVSV-LASV-G and VSV-EBOV-G were able to cross the blood brain barrier andselectively infect brain tumors with little or no infection of normalneurons or glia, and no adverse effects. VSV-LASV-G completelyeliminated brain tumors and prolonged the lives of tumor-bearing miceindefinitely. VSV containing the Ebola glycoprotein also crossed theblood brain barrier and targeted the brain tumor, but showed onlypartial infection of the glioma.

VSV neurotoxicity can be reduced by generating peripheral immunity inadvance of intracerebral inoculation (Ozduman, et al., J. Virol.,83:11540-11549 (2009) or by administering exogenous type 1 interferon,or via intra cerebral viral vectors that generate interferon (Wollmann,et al., Virology, in press. (2014). Attenuated VSVs have beenconstructed by a number of molecular alterations, including reduction ofcytoplasmic amino acids in the G protein, mutations in the M geneparticularly at M51, adding genes upstream of viral genes (Roberts, etal., J. Virol., 73:3723-3732 (1999); Ahmed, et al., Virology, 33:34-39(2004); Ramsburg, et al., J. Virol., 79:15043-15053 (2005)), but most ofthese resultant VSVs still retain negative side effects in the brain dueto neuronal infection. Even a VSV currently in clinical tests thatexpresses interferon (VSV-IFN), although attenuated, is problematic andcan be lethal if it gains access to the brain. None of these recombinantstrategies directly eliminate the lethal neurotropism of the viruswithin the brain conferred by the VSV-G protein. In contrast, all five Gprotein chimeric viruses used in the disclosed studies showed reducedneuron infection, and the two tested in vivo showed complete safetywithin the brain. Even substitution of the glycoprotein from rabies, avirus with well-known neuronal targeting, although it still showed anunacceptably high neuron targeting, still showed reduced infection ofneurons compared with the VSV glycoprotein, and has been shownrelatively safe in the brain (Beier, et al., Proc. Natl. Acad. Sci. USA,108:15414-15419 (2011), underlining the importance of eliminating theVSV glycoprotein.

Neither VSV-EBOV-G nor VSV-LASV-G injected intracerebrally into normalmice, immunocompromised SCID mice, or rats evoked any adverse action,whereas similar concentrations of other VSVs with the native G,including attenuated VSV-CT9-M51 (Wollmann, et al., Virology, in press.(2014), VSV-1′ GFP, VSVmIFNα, and VSV-M51 (FIG. 2A-2B) were lethal.

This safety is also corroborated with previous studies that have usedsimilar virus recombinants with Lassa (or Ebola) glycoproteins in placeof the VSV G-protein as immunization vehicles to protect againstwild-type Lassa or Ebola viruses; and deletion of genes other that theglycoprotein gene eliminates the toxicity and disease potentialassociated with wild type Ebola and Lassa. Lassa and Ebola virus mayhave arisen from a common ancestor virus (Gallaher, et al., BMCMicrobial., 1:1, 6 pages, (2001)). VSV-LASV-G was previously shown safein rodents after intraperitoneal injection, but intracranial safety wasnot investigated (Garbutt, et al., J. Viral., 78:5458-5465 (2004). Onehuman has been injected with Ebola-VSV as a vaccine after a labaccident, with no adverse consequence (Gunther, et al., J. Infect. Dis.,204 Suppl 3:S785-S790 (2011)). The results disclosed herein show thatboth VSV-LASV-G and VSV-EBOV-G are safe in the rodent brain. The GFPreporter gene in both VSV-LASV-G and VSV-wtG provides some attenuationto both viruses by reducing expression of other VSV genes; in spite ofthis, VSV with the wild type glycoprotein was still lethal within thebrain even when carrying the attenuating GFP gene, whereas VSV-LASV-Gand -EBOV-G were safe in the brain indicating that safety in the brainis due to the absence of the neurotoxic VSV glycoprotein. This view issupported by the data showing safety of VSV-LASV in chimeric viruses notcontaining the GFP gene. The lack of VSV-LASV-G infection of neurons inthe rodent brain parallels the in vitro studies with human neuronsshowing a lack of infection, and indicating that the virus may also besafe in the human brain. This is consistent with the findings which didnot consider the oncolytic potential, but did show that these chimericviruses evoked a strong humoral and cellular immune response, andimportantly, evoked no adverse health consequences, even when injecteddirectly into the brains of rodents or non-human primates (Geisbert, etal., PLoS Med, 2:e183 (2005); Geisbert, et al., PLoS Pathog., 4:e1000225(2008); Geisbert, et al., J Virol., 83(14):7296-7304 (2009); Mire, etal., PLoS Negl. Trop Dis, 6:e1567 (2012)). Initiation of a strong immuneresponse is another benefit, and can generate a secondary immune attackon the brain tumor.

Such strong attenuation of virus infection on normal cells might alsolead to lack of efficiency in tumor destruction. Lassa-VSV wasnonetheless capable of selectively infecting and killing GBM cells invitro and in the mouse brain after intravenous or intracerebral virusadministration, and substantially prolonged cancer survival far beyondthat of control tumor-bearing mice that received no virus. In fact, atthe time when all tumor bearing control mice had died from the expandingbrain tumor, none of the mice in which tumors were treated withVSV-Lassa showed any obvious symptoms from the tumor, or from the virus.

In conclusion, other VSVs, even those attenuated, can lead to adverseneurological consequences or death. In contrast, Lassa-VSV shows nodetectable adverse effects when injected directly into the brain.

Example 3 IFN has Little Effect in Attenuating Infection of Gliomas byVSV-LASV-G Materials and Methods

Quantitative RT-PCR

Mouse neuronal cultures and human U87 glioma cells were cultured in6-well plates. VSV-LASV-G or VSV-wtG was added at an MOI of 1 andcultures were incubated for 20 minutes at 4° C. to test virus binding orfor 30 minutes at 37° C. to test virus binding+internalization,respectively, as described elsewhere (Ozduman et al, 2008). Experimentswere performed in duplicate. Cells were washed five times with PBS priorto RNA isolation using TRIzol Reagent (Invitrogen, Carlsbad, Calif.).Stratascript reverse transcriptase kit (Stratagene) was used for cDNAgeneration. TaqMan gene expression assays (Applied Biosystems, FosterCity, Calif.) were used to quantify the expression of β-actin and VSVgenomes using an ICycler iQ Real time PCR system (Bio-Rad, Hercules,Calif.). For specific VSV genome detection (excluding viral mRNA)primers were designed to yield a product that spanned the junctionbetween N and P genes. PCR samples were measured in triplicates,normalized to β-actin expression and compared to expression of VSVLASV-Gbinding to neurons as reference (ΔΔt method).

Results

The results described herein show that the identity of the G protein hasa profound effect on neuronal toxicity in the context of chimeric VSV,with viruses bearing LASV-G or LCMV-G being the least neurotropic. Toelucidate mechanisms whereby the chimeric viruses appeared to showstrong infection of glioma, but less infection of normal neurons andglia, the innate immune response was examined. In contrast to theprotective effect of IFN on cultures of normal human neurons, glia, orfibroblasts, microscopy reveled that IFN had little effect inattenuating infection by VSV-LASV-G of U118 and U87 human gliomas, asvirtually all glioma cells were infected and GFP-positive by 24 hourspost-inoculation (FIG. 3A).

These data are consistent with the view that a primary mechanismunderlying the selective VSV-LASV-G infection of cancer cells overnormal glia is related to deficiencies in innate immunity. The lack ofprotection by added IFN points to a deficient IFN response amongVSV-LASV-G susceptible tumors, similar to the mechanism described forthe enhanced relative susceptibility of a number of tumor types tonative VSV and to several other viruses including Newcastle diseasevirus, reovirus and myxoma virus (Phuangsab, et al., Cancer Lett.,172:27-36 (2001); Strong, et al., EMBO J., 17:3351-3362 (1998); Wang, etal., Nature Immunol., 5:1266-1274 (2004)). The observation that normalbrain cells in SCID mice which are deficient in T- and B-cell antiviraldefenses showed little infection and no adverse effects from VSV-LASV-Gin the brain further supports the view that an innate immune mechanismis protective of normal brain cells.

To test whether the intrinsic IFN system is important for the resistanceof the brain to VSV-LASV-G, VSV-LASV-G was injected into the brains ofmice (n=5) lacking the type I IFN receptor. Although IFNα/β-R−/− micewith intracerebral infection from VSV-LASV-G survived longer than thosewith VSV-wtG (n=3) (FIG. 3B), all these mice ultimately died within aweek of CNS inoculation. These data support the view that an innateIFN-mediated immune response is important for both short and long termsurvival after VSV-LASV-G infection of the brain.

This motivated a comparison of binding of VSV-wtG and VSV-LASV-G toglioma and neurons. As judged by quantitative RT-PCR, both viruses boundsimilarly well to glioma cells and to neurons at 4° C. (FIG. 3C), atemperature at which endocytosis is inhibited. At 37° C., approximately2-logs more virus becomes cell-associated than at 4° C., indicatingeffective and similar rates of internalization in both cell types andfor both viruses.

Next, the ability of infected glioma cells and neurons to generateprogeny virions was examined. On gliomas, both VSV-wtG (FIG. 3D) andVSV-LASV-G (FIG. 3E) show substantial virus replication. In contrast,replication of VSV-LASV-G was greatly attenuated in neurons, by 4.5 logsrelative to VSV-wtG which showed robust replication in neuron cultures(FIG. 3D-3E). Together, these data indicate that although both VSV-wtGand VSV-LASV-G bind to glioma cells and neurons, VSV-LASV-G replicatespoorly in neurons. In neurons, a block to replication appears mostlikely to occur at the point of endosomal escape and uncoating, which isG-protein mediated, or possibly a subsequent step.

These data support a conclusion that the advantage of the Lassa virusglycoprotein is that it produces a selective block to replication inneurons but not in a wide variety of human tumors. This block to neuroninfection appears not to be at the binding or internalization steps butat a step further downstream in the life cycle, possibly at theuncoating/endosomal escape step in neurons, given the importance of theglycoprotein to this process (Rigaut, et al., J. Virol., 65:2622-2628(1991).

Type 1 IFN is important to the selectivity and safety of VSV-LASV; micelacking IFN receptor succumbed to virus inoculation. Previous reportsindicate that whereas type 2 and 3 IFN may also contribute to immunityin the brain (van den Pol, et al., J. Virol., 88:3695-3704 (2014), type1 IFN is necessary for survival. The impairment of innate immunitycharacteristic of ontogenically transformed cells allows VSV-LASV toreplicate rapidly, with cytolytic consequences for tumor cells. Additionof IFN to glioma had little to no effect on the infectivity of VSV-LASV,but provided additional protection to normal neurons and glia. Theexquisite tumor-selectivity of VSV-LASV is thus a consequence of bothreduced neurotropsism (via substitution of the Lassa G for the VSVG-protein) and virus susceptibility to IFN in normal cells; thus thevirus is able to very selectively infect and destroy tumors whetherentering from the blood stream into the brain, or crossing from aninfected tumor to infect a distant locus of tumor cell growth in thecontralateral brain, all without infecting the intervening healthy braintissue. The fact that the innate immune system appears able to containVSV-LASV-G indicates that the virus may prove safe to treat theincreased incidence of cancers found in patients with compromisedadaptive immune systems, for instance with AIDS.

Example 4 The Chimeric Virus Lassa-VSV Selectively Infects and KillsGlioma Materials and Methods

Migration Assays

Red fluorescence marker RFP expressing tumor cells (U87 humanglioblastoma cells and YUMAC human melanoma cells, respectively) wereimplanted into mice: two tumors per mouse—one in the left striatum, anda second in the right striatum.

14 days after tumor placement, Lassa-VSV expressing a green reportergene GFP was then injected into the right tumor only.

Eight days later, mice were euthanized and perfused transcardially with4% paraformaldehyde. Brains were harvested and analyzed for tumorexpansion and virus infection using red and green fluorescence on astereomicroscope for whole brain analysis and on an Olympus microscopefor analysis of brain sections.

Results

The ability of chimeric virus to target and destroy brain tumors wasalso tested. Fifteen days after implant of red fluorescent human gliomainto the SCID mouse brain, VSV-LASV-G (10⁶ pfu in 100 ul) was injectedintravenously (tail vein). This resulted in highly selective tumorinfection and complete destruction of tumor cells within the brain. Micewith brain tumors survived only if treated with VSVLASV-G, with noadverse effect as of 80 days (FIG. 4). Mice with tumors that did notreceive virus all died from the tumor (median 29 day survival; n=8; FIG.4). The chimeric virus VSV-EBOV-G also targeted brain tumors afterintravenous inoculation (FIG. 4); VSV-EBOV-G extended life minimally(median survival 34 days; n=8) (FIG. 4).

Histological analysis showed large tumors in mice not treated withvirus, and incomplete but selective infection of the tumor treated withVSV-EBOV-G. In contrast, few tumor cells if any were found followingtreatment with VSV-LASV-G, indicating highly selective infection anddestruction of glioma.

In another short term experiment, mice showed near-complete infection ofbilateral tumor masses 8 days post-inoculation, with little infectionoutside the tumor area indicating a rapid and selective VSV-LASV-Ganti-tumor action (n=4).

Using a syngeneic rodent tumor model, the ability of VSV-LASV-G toinfect rat glioma in immunocompetent rats was tested. VSV-LASV-G showedselective strong cytolytic infection of CNS-1 glioma tumors 3 days afterintracerebral inoculation, with little detectable infection of normalbrain, demonstrating the VSV-LASV-G selectively infects glioma not onlyin mice, but also in immunocompetent rats.

A major problem with gliomas is tumor cell migration within the brain.Thus skilled neurosurgeons can debulk the tumor, but cannot eliminatemigrating cells or tumors that arise from migrating cells. To modelthis, human glioma were implanted in the left and right side of the SCIDmouse brain with (in striatum). Fifteen days later VSV-LASV-G wasstereotactically injected unilaterally, only into the tumor on the rightside. Eight days later, the brains were examined. VSV-LASV-G hadcompletely destroyed the inoculated tumor on the right side of thebrain, and the virus had migrated to the contralateral left tumor andbegun the process of infection and destruction without infecting theintervening normal brain. Accordingly, in addition to completelydestroying the right tumor, Lassa-VSV migrated within the brain, and hadbegun to infect and kill the left tumor without detectable infection ofnormal brain cells surrounding the tumors. Remarkably, VSV-LASV-Gselectively destroyed the brain tumor with no adverse effects to theSCID mouse brain. These observations indicate that the virus wassuppressed in the brain by a T- and B-cell-independent mechanism.

This is exactly the type of phenotype needed, that is, a virus that caninfect multiple tumors in different CNS sites with no collateral damageto normal brain. Peripheral immunization has been shown to protect thebrain from intracranial VSV (Ozduman, et al., J Virology, 83:11540-11549(2009)). Even without immunization, Lassa-VSV showed no neurotoxicity,whereas other VSVs did show intracranial neurotoxicity. Lassa-VSV wassubstantially more attenuated than the most attenuated VSV used(Ozduman, et al., J. Virology, 83:11540-11549 (2009); Wollmann, et al.,J. Virol, 84:1563-73 (2010)), underlining its safety in the brain.

One potential limitation of VSVs with point and some other types ofmutations (VSV-M51, VSV1′2′-GFP, and others) is that additionalspontaneous mutations can cause the virus to revert back to a moreaggressive wild type virus. That is unlikely in Lassa-VSV, as there isno mechanism whereby the Lassa-glycoprotein can mutate into the VSVglycoprotein, or where the VSV G-protein can be reconstituted, and alsono way in which the non-glycoprotein genes of Lassa could appear in therecombinant virus.

Example 5 The Chimeric Virus Lassa-VSV Infects and Kills Melanoma

Next experiments were designed to determine if VSV-LASV-G is selectivefor gliomas, or is able to target other types of metastatic braincancer. Melanomas are the deadliest form of skin cancer, and one of thechief problems is metastasis into the brain (Carlin, et al., Cancer J,18:208-212 (2012). Red fluorescent human melanoma was injected into theleft and right side of the brain, similar to the glioma experimentsdescribed above. VSV-LASV-G was subsequently injected unilaterally intothe right side melanoma. VSV-LASV-G not only caused complete destructionof the injected tumor mass, it also diffused across the brain midline,and showed strong infection of the non-injected contralateral melanoma,with no neuron infection in the intervening (SCID) brain.

Because VSV-LASV-G infected two unrelated brain tumor types, glioma andmelanoma, it was also tested on other types of human cancer cells.VSV-LASV-G infected and replicated in colon, prostate, breast, bone, andbladder cancer cells, indicating its oncolytic potential was notrestricted to glioma and melanoma brain tumors.

These results indicate that VSV-LASV-G is effective not only againstglioma (which arise within the brain) but also against melanoma, acancer that arises in the skin but metastasizes into the brain resultingin death within a few months of entering the brain. There is also astrong viral tropism for other types of human cancer cells includingprostate, breast, colon, and bladder which can sometimes metastasizeinto the brain. The broad infectivity and cytolysis of multiple types ofcancer cells indicate that VSV-LASV-G may also be effective in targetingother types of brain tumors not tested here, including meningioma,astrocytoma, ependymoma, and oligodendroglioma. The elimination ofneurotropism by substitution of the Lassa glycoprotein for the VSVglycoprotein would provide an increased level of brain safety even ifthe virus were used to attack peripheral cancers.

In vitro testing indicates VSV-LASV had the best combination of reducedneurotropism and broad infectivity across different gliomas. VSV-EBOValso demonstrated broad glioma infectivity in vitro; although VSV-EBOVtargeted tumors in the brain after intravenous application, the viruswas not very effective in destroying the tumor. VSV-LCMV had a reducedneurotropism similar to VSV-LASV, but did not show as great or broad apotential for infectivity of gliomas, and formed smaller plaques thanVSV-LASV in the human gliomas tested.

Example 6 VSV-LASV-G Evokes a Humoral Immune Response

To test whether VSV-LASV-G would evoke a humoral immune response, micewere inoculated intranasal and intramuscularly with this virus.VSV-LASV-G generated high titer antisera to VSV-LASV-G infected cellsand also to the GFP transgene. Antisera with dilutions out to 1:10,000generated positive immunostaining on GFP-expressing transgenic mice,indicating the potential for a secondary systemic immune responseagainst VSV-LASV-G infected gliomas as reported for wildtype VSVs(Publicover, et al., J. Virol., 78:9317-9324 (2004). It is interestingto note that chimeric VSV-LCMV/G was shown to be only weakly immunogenicwith regard to generation of neutralizing antibodies against the virus(Muik, et al., Cancer Res., 74:3567-3578 (2014). VSV-LASV-G appeared tocompletely eliminate human brain tumors in these experiments; VSVs alsogenerated a strong immune response against tumors which could serve toaugment tumor eradication in the event of incomplete direct tumordestruction (Wongthida, et al., Hum. Gene Ther., 22:1343-1353 (2011).Chimeric VSVs that generate a strong immune response may also bebeneficial in terms of evoking a strong secondary immune responseagainst tumor related antigens.

VSV is a very promising vaccine platform, and even single inoculationsgenerate strong cellular and humoral immunity (Publicover, et al, J.Virol., 79:13231-13238 (2005); Buonocore, et al., J. Virol.,76:6865-6872 (2002); Schell, et al., J. Virol., 85:5764-5772 (2011)),but would advance further with the risk of neurotoxicity effectivelyeliminated, for example by substitution of the Lassa glycoprotein forthe VSV glycoprotein. VSV-LASV-G may prove beneficial as a vaccinevector, given its potent immunogenicity. Immunogenic viral vectors couldbe generated against other pathogenic organisms by substituting a genecoding for a protein from the pathogenic microbe in place of the GFPgene within VSV-LASV-G; use of the Lassa glycoprotein in place of theVSV glycoprotein could provide a safer vector for immunization thanwildtype VSV.

Example 7 The Chimeric Virus Lassa-VSV Protects from Glioma Materialsand Methods

Three groups of SCID mice received human glioma implants of similarsize. 15 days later, one group received Lassa-VSV intravenously (tailvein), the second group received Ebola-VSV intravenously, and the thirdgroup served as a tumor-only control. Mice were monitored for changes inbody weight. Mice showing body weight <75% of pre-tumor body weight wereeuthanized, as per animal use regulations.

Results

A mouse weight graph (FIG. 5) showed that mice receiving Lassa-VSVshowed no strong reduction in body weight, whereas control mice withtumors show body weight reduction from the expanding tumors. Mice withtumors inoculated with Ebola-VSV (VSV-Eb-G) showed a reduced body weightfrom the expanding tumor. Lassa-VSV is safe in the brain, as micemaintained a normal body weight.

A mouse survival graph (FIG. 4) showed that Lassa-VSV protected micefrom an implanted glioma. In contrast, control mice with the same tumorshowed a median survival of 29 days post-glioma injection. All controlswere dead by day 33 after tumor implant. The Ebola-VSV gave someprotection against the tumor with a median survival of 34 dayspost-glioma implant, but ultimately, all were dead by 57 days aftertumor implant. All mice treated with Lassa-VSV survived >43 days afterLassa-VSV inoculation, and >80 days after glioma implant. The increasedlongevity of tumor bearing mice receiving Lassa-VSV was statisticallysignificant (p<0.01; n=8 in each group). Ultimately all controls diedfrom the glioma. In striking contrast, Lassa-VSV crossed the blood brainbarrier, infected and destroyed the tumor, and these mice all survived.Upon histological examination after euthanasia, large red gliomas werefound in control mice, but were absent from Lassa-VSV-treated mice.Furthermore, since these SCID mice had severely attenuated systemicimmune system (necessary for implantation of human glioma), but stillsurvived peripheral and CNS infection with the virus, the chimericLassa-VSV is remarkably safe within the body, and particularly withinthe brain.

When injected into a brain tumor, Lassa-VSV kills tumor cells, andprolongs life with no detectable neurological consequences. Wheninjected intravenously, the virus crosses the blood brain barrier andselectively infects gliomas.

1. A method of treating cancer comprising administering to a subjectwith cancer a pharmaceutical composition comprising an effective amountof a chimeric Vesicular stomatitis Indiana virus (VSV) virus to treatthe cancer, wherein the chimeric VSV virus comprises a VSV backgroundwith one or more heterologous viral glycoproteins in place of the VSVG-protein, wherein the G protein is not from Lymphocyticchoriomeningitis (LCMV).
 2. A method of reducing one or more symptoms ofcancer in a subject comprising administering to a subject with cancer apharmaceutical composition comprising an effective amount of a chimericVSV virus to reduce one or more symptoms of the cancer in the subject,wherein the chimeric VSV virus comprises a VSV background with one ormore heterologous viral glycoproteins in place of the VSV G-protein,wherein the G protein is not from LCMV.
 3. The method of claim 1 whereinthe one or more heterologous viral glycoproteins is a Lassa virusglycoprotein precursor (GPC), a functional fragment or variant thereof,or an Ebola glycoprotein, a variant or functional fragment thereof. 4.The method of claim 3 wherein the one or more heterologous viralglycoproteins is Lassa virus GP1, Lassa virus GP2, or a combinationthereof.
 5. The method of claim 1 wherein the VSV background is VSVIndiana, VSV New Jersey, VSV Alagoas, (formerly Indiana 3), VSV Cocal(formerly Indiana 2), VSV Chandipura, VSV Isfahan, VSV San Juan, VSVOrsay, VSV Glasgow, or a recombinant VSV comprising at least 1 gene fromtwo or more VSV strains or serotypes selected from the group consistingof VSV Indiana, VSV New Jersey, VSV Alagoas, (formerly Indiana 3), VSVCocal (formerly Indiana 2), VSV Chandipura, VSV Isfahan, VSV San Juan,VSV Orsay, and VSV Glasgow.
 6. The method of claim 1 wherein theheterologous glycoprotein is from Lassa virus strain Josiah.
 7. Themethod of claim 1 wherein the chimeric VSV virus further comprises oneor more additional heterologous proteins.
 8. The method of claim 7wherein the chimeric VSV virus's genome encodes the one or moreheterologous genes.
 9. The method of claim 7 wherein the one or moreadditional heterologous proteins is a therapeutic protein, a reporter, avaccine antigen, a targeting moiety, or a combination thereof.
 10. Themethod of claim 9 wherein the one or more additional heterologousproteins includes at least one therapeutic protein selected from thegroup consisting of IL-28, IL-2, FLT3L, GM-CSF, IL-4,IL-7, IL-12, andTRAIL.
 11. The method of claim 9 wherein the one or more additionalheterologous proteins includes at least one reporter selected from thegroup consisting of green florescent protein, red florescent protein,carcinoembryonic antigen, secreted alkaline phosphatase, and the betasubunit of chorionic gonadotropin.
 12. The method of claim 1 wherein thecancer is selected from the group consisting of multiple myeloma, bone,bladder, brain, breast, cervical, colo-rectal, esophageal, kidney,liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, anduterine.
 13. The method of claim 12 wherein the brain cancer isoligodendroglioma, meningioma, supratentorial ependymona, pineal regiontumors, medulloblastoma, cerebellar astrocytoma, infratentorialependymona, brainstem glioma, schwannomas, pituitary tumors,craniopharyngioma, optic glioma, and astrocytoma.
 14. The method ofclaim 12 wherein the brain cancer is glioblastoma.
 15. The method ofclaim 1 wherein the virus is in a dosage of between 10² and 10¹² PFU.16. The method of claim 1 wherein the pharmaceutical composition isadministered locally to the site of the cancer.
 17. The method of claim16 wherein the pharmaceutical composition is injected into or adjacentto a tumor in the subject.
 18. The method of claim 1 wherein thepharmaceutical composition is administered systemically to the subject.19. The method of claim 18 wherein the pharmaceutical composition isadministered by intravenous injection or infusion.
 20. The method ofclaim 1 wherein the pharmaceutical composition is administered to thesubject intranasally or by pulmonary delivery.
 21. The method of claim 1further comprising administering to the subject a second therapeuticagent.
 22. The method of claim 21 wherein the second therapeutic agentis an anticancer agent, a therapeutic protein, or an immunosuppressant.23. The method of claim 22 wherein immunosuppressant is a histonedeacetylase (HDAC) inhibitor or an interferon blocker.
 24. The method ofclaim 23 wherein the second therapeutic agent is selected from the groupconsisting of valproate, the vacccinia protein B18R, Jak inhibitor 1,and vorinostat.
 25. The method of claim 1 wherein the pharmaceuticalcomposition is administered in combination with surgery.
 26. The methodof claim 25 wherein the surgery is prior to administration of thepharmaceutical composition.
 27. The method of claim 1 further comprisingadministering the subject an immunizing composition comprising a viruseffective to immunize to the subject to the chimeric VSV virus prior toadministration of the pharmaceutical composition.
 28. The method ofclaim 27 wherein the virus of the immunizing composition is the chimericVSV virus.
 29. The method of claim 27 wherein the immunizing compositionis administered to the subject days, weeks, or month beforeadministration of the pharmaceutical composition.
 30. The method ofclaim 1 where the chimeric virus has a higher oncolytic potential thanthe parent virus of the chimeric VSV wherein the G protein is notreplaced with a heterologous glycoprotein.
 31. The method of claim 1wherein the chimeric VSV shows little or no ability to infect normal orhealth neurons.
 32. A method of treating a subject for cancer comprising(a) infecting isolated cancer cells with an effective amount of achimeric VSV virus (b) administrating the infected cells to the subjectin an effective amount to induce an immune response against the cancercells in the subject; wherein the chimeric VSV virus comprises a VSVbackground with one or more heterologous viral glycoproteins in place ofthe VSV G-protein, wherein the G protein is not from Lymphocyticchoriomeningitis (LCMV).
 33. The method of claim 32 further comprisingirradiating the cells to prevent their proliferation.
 34. The method ofclaim 32 wherein the subject has cancer.
 35. The method of claim 32wherein the subject does not have cancer.
 36. A method of preparingcells for adaptive T cell therapy comprising administering to a subjectwith cancer a pharmaceutical composition comprising an effective amountof a chimeric VSV virus to increase the number of cytotoxic T cells(CTL) which can directly kill the cancer, wherein the chimeric VSV viruscomprises a VSV background with one or more heterologous viralglycoproteins in place of the VSV G-protein, wherein the G protein isnot from Lymphocytic choriomeningitis (LCMV).
 37. A method of preparingcells for adaptive T cell therapy comprising administering to a subjectwith cancer a pharmaceutical composition comprising an effective amountof a chimeric VSV virus to increase the number of CD4+ T cells which candirect an immune response against the cancer, wherein the chimeric VSVvirus comprises a VSV background with one or more heterologous viralglycoproteins in place of the VSV G-protein, wherein the G protein isnot from Lymphocytic choriomeningitis (LCMV).
 38. The method of claim 36wherein the one or more heterologous viral glycoproteins is Lassa virusGP1, Lassa virus GP2, or a combination thereof.
 39. The method of claim36 wherein the cancer is a brain cancer or cancer that can metastasizeinto the brain.
 40. The method of claim 39 wherein the brain cancer isglioblastoma.
 41. The method of claim 39 wherein the cancer that canmetastasize into the brain is selected from the group consisting of lungcancer, breast cancer, and melanoma.
 42. The method of claim 36 whereinthe cancer is not a brain cancer.
 43. The method of claim 37 comprisingisolating the T cells, propagating them in vitro, and administering themto a subject in need thereof.
 44. A pharmaceutical dosage unitcomprising an effective amount of a chimeric VSV virus to treat cancerin subject in need thereof, wherein the chimeric VSV virus comprises aVSV background with one or more heterologous viral glycoproteins inplace of the VSV G-protein, wherein the G protein is not fromLymphocytic choriomeningitis (LCMV).
 45. The pharmaceutical dosage unitof claim 44 wherein the one or more heterologous viral glycoproteins isLassa virus GP1, Lassa virus GP2, or a combination thereof.
 46. A kitcomprising the pharmaceutical dosage unit of claim 44 in a sterile vial.47. A method of vaccinating a subject against an antigen comprisingadministering the subject an effective amount of a chimeric VSV viruscomprising a VSV background with one or more heterologous viralglycoproteins in place of the VSV G-protein and an antigen, in aneffective amount to enhance an immune response against the antigen,wherein the G protein is not from Lymphocytic choriomeningitis (LCMV).48. The method of claim 47, wherein the G-protein is a Lassa virus Gprotein.
 49. The method of claim 48 wherein the antigen is a non-Lassavirus, non-VSV-virus protein or polypeptide derived from a bacterium, aparasite, a plant, a protozoan, a fungus, or another virus, or is acancer antigen.
 50. The method of claim 48 wherein the antigen isderived from influenza, HIV, polio, or measles.